RhoA Activation Research Articles

RhoGDI in RBL-2H3 cells acts as a negative regulator of Rho GTPase signaling to inhibit granule exocytosis.

Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however, the proteins that regulate Rho GTPases during this process are not well defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2, and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown (DKD) strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 DKD strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol.

Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity.

Adaptive immunity relies on dendritic cell (DC) migration to transport antigens from tissues to lymph nodes. Galectins, a family of β-galactoside-binding proteins, control cell membrane organisation, exerting crucial roles in multiple physiological processes. Here, we report a novel mechanism underlying cell polarity and uropod retraction. We demonstrate that galectin-9 regulates chemokine-driven and basal DC migration both in humans and mice, indicating a conserved function for this lectin. We identified the underlying mechanism, namely a deficiency in cell rear contractility mediated by galectin-9 interaction with CD44 that in turn regulates RhoA activity. Analysis of DC motility in the 3D tumour-microenvironment revealed galectin-9 is also required for DC infiltration. Moreover, exogenous galectin-9 rescued the motility of tumour-immunocompromised human blood DCs, validating the physiological relevance of galectin-9 in DC migration and underscoring its implications for DC-based immunotherapies. Our results identify galectin-9 as a necessary mechanistic component for DC motility and highlight a novel role for the lectin in regulating cell polarity and contractility.

Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling.

Barrier function is critical for epithelial tissues. Epithelial cells maintain barrier function via tight junctions, which must be remodeled to allow for cell- and tissue-scale shape changes. How barrier function is maintained and remodeled as epithelial cells change shape remains unclear.The scaffolding protein Anillin is required for generating effective actomyosin contraction to reinforce damaged tight junctions; lack of reinforcement leads to repeated barrier leaks.These findings highlight a novel role for Anillin in tight junction remodeling and suggest that Anillin's ability to tune the level and duration of local Rho activation affects the contractile output.

FRET verification of crucial interaction sites in RhoA regulation mediated by RhoGDI

The small GTPase Rho family are the major factors in mediating actin cytoskeleton dynamics. Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) serve as important negative regulators by complexing with inactive Rho into the cytoplasm. However, how these two molecules interact still needs experimental verification. Based on fluorescence resonance energy transfer (FRET) measurements, we would demonstrate crucial sites in RhoGDI and RhoA for this regulatory role. Cotransfection of RhoGDI markedly reduced RhoA or Cdc42 activity in airway smooth muscle (ASM) cells, while D185R-RhoGDI mutant reversed this decrease, indicating that RhoGDI Asp185 residue is essential for the molecular interaction. R68D-RhoA (mutation in the switch II region) resulted in a deficiency in RhoGDI regulation, while TV37/38NG-RhoA (in the switch I region) displayed low RhoA activity. Hence, the Arg68 site in RhoA is indispensable for regulation by RhoGDI, and Thr37Val38 site is important for maintaining RhoA activity. Additionally, microtubule but not actin cytoskeleton showed inhibitory role in RhoA activity, while the dissolution of either cytoskeleton did not change the regulatory role of RhoGDI. In checking the downstream effect, reduction of RhoA activity induced by PDGF stimulation or RhoGDI decreased cellular stress fibers. In this study, FRET visualization was applied to have experimentally demonstrated the interaction sites and crucial role of RhoGDI in regulating RhoA activity.Graphical

Investigating the Correlations between Patient Demographics and Complications Following Total Knee Arthroplasty: A Cross-Sectional Study

Introduction: This study investigates the relationship between patient age, gender, and various complications, such as pain, restricted range of motion (ROM), and hemorrhage following total knee arthroplasty. Materials and Methods: This cross-sectional study included 62 patients (Mean±SD age= 65.9±7.38 years; female participants=74.2%) who underwent total knee arthroplasty at Atieh Hospital in Tehran, Iran, between 2020 and 2021. Pain levels were assessed using the numerical pain rating score. Meanwhile, knee joint ROM was evaluated using a reliable mobile application. Hemorrhage was measured through knee joint drainage. Statistical analyses were performed to analyze the data. Results: Women reported higher pain levels and more restricted ROM in passive knee extension and active/passive knee flexion compared to men (P<0.05). However, no significant differences were observed between genders in active knee extension and hemorrhage. A significant inverse correlation was found between pain and ROM in passive knee extension (rho=-0.41, P≤0.001), as well as in active (rho=-0.5, P≤0.001) and passive knee flexion (rho= -0.59, P≤0.001). However, no significant relationship was observed between pain and active knee extension or hemorrhage. Age showed no statistically significant correlation with the other variables. Conclusion: This study highlights the association between higher pain levels and greater ROM restrictions in passive knee extension and active/passive knee flexion following total knee arthroplasty; however, patient age and gender were not found to be associated with postoperative hemorrhage. These findings underscore the importance of closely monitoring pain and ROM, particularly in women undergoing total knee arthroplasty.

PDGFRB promotes dedifferentiation and pulmonary metastasis through rearrangement of cytoskeleton under hypoxic microenvironment in osteosarcoma

BackgroundOsteosarcoma (OS) cells commonly suffer from hypoxia and dedifferentiation, resulting in poor prognosis. We plan to identify the role of hypoxia on dedifferentiation and the associated cellular signaling. MethodsWe performed sphere formation assays and determined spheroid cells as dedifferentiated cells by detecting stem cell-like markers. RNAi assay was used to explore the relationship between hypoxia inducible factor 1 subunit alpha (HIF1A) and platelet derived growth factor receptor beta (PDGFRB). We obtained PDGFRB knockdown and overexpression cells through lentiviral infection experiments and detected the expression of PDGFRB, p-PDGFRB, focal adhesion kinase (FAK), p-FAK, phosphorylated myosin light chain 2 (p-MLC2), and ras homolog family member A (RhoA) in each group. The effects of PDGFRB on cytoskeleton rearrangement and cell adhesion were explored by immunocytochemistry. Wound-healing experiments, transwell assays, and animal trials were employed to investigate the effect of PDGFRB on OS cell metastasis both in vitro and in vivo. ResultsDedifferentiated OS cells were found to exhibit high expression of HIF1A and PDGFRB, and HIF1A upregulated PDGFRB, subsequently activated RhoA, and increased the phosphorylation of MLC2. PDGFRB also enhanced the phosphorylation of FAK. The OS cell morphology and vinculin distribution were altered by PDGFRB. PDGFRB promoted cell dedifferentiation and had a significant impact on the migration and invasion abilities of OS cells in vitro. In addition, PDGFRB increased pulmonary metastasis of OS cells in vivo. ConclusionOur results demonstrated that HIF1A up-regulated PDGFRB under hypoxic conditions, and PDGFRB regulated the actin cytoskeleton, a process likely linked to the activation of RhoA and the phosphorylation of, thereby promoting OS dedifferentiation and pulmonary metastasis.

A nonactivating ITGB3 mutation in the β3 cytoplasmic region causes macrothrombocytopenia with an impaired αIIbβ3/RhoA pathway

AbstractAlmost all mutations of ITGA2B or ITGB3 identified in congenital macrothrombocytopenia induce constitutive activation of αIIbβ3. However, whether concomitant αIIbβ3 activation is essential for macrothrombocytopenia development remains unknown. Recently, we identified the β3(R760C) mutation that does not induce αIIbβ3 activation in a patient with macrothrombocytopenia. The family study showed that macrothrombocytopenia with reduced expression of αIIbβ3 and glycoprotein IV (GPVI) appeared to be associated with patients heterozygous for β3(R760C). We generated β3(R760C) knockin (KI) mice and investigated the effects of the mutation on platelet/megakaryote biology. Macrothrombocytopenia was decreased to 76% and 40% of platelet counts in heterozygous (Hetero) and homozygous (Homo) KI mice, respectively, when compared with the wild-type mice. Platelet αIIbβ3 and GPVI expression were decreased in KI mice, and αIIbβ3 activation was not detected in nonstimulated KI platelets. Thus, the hetero KI mice reproduced the phenotype of the human participant, indicating that the β3(R760C) mutation is responsible for the macrothrombocytopenia. Platelet aggregation, agonist-induced JON/A binding, and P-selectin expression were impaired in KI mice. Platelet spreading on fibrinogen was also impaired in Homo mice with adenosine 5′-diphosphate or thrombin stimulation. Filopodia and lamellipodia formation was impaired in fibrinogen-adhered megakaryocytes of Homo mice with significantly impaired RhoA activation. Proplatelet formation in Homo mice was impaired with abnormal morphology. In addition, platelet life span was shortened in Homo mice. These data indicate that the β3(R760C) mutation impairs the inside-out and outside-in signaling of αIIbβ3, and abnormal actin rearrangement and impaired RhoA activation may play major roles in macrothrombocytopenia.

The CYK-4 GAP domain regulates cortical targeting of centralspindlin to promote contractile ring assembly and facilitate ring dissolution.

During cytokinesis, an equatorial contractile ring partitions the cell contents. Contractile ring assembly requires an equatorial zone of active GTP-bound RhoA generated by the guanine nucleotide exchange factor ECT21,2. ECT2 is activated by centralspindlin, a complex composed of two molecules each of kinesin-6 and CYK4. During anaphase, Centralspindlin is activated at the central spindle between the separating chromosomes and diffuses to the plasma membrane, where it engages with ECT2 via its N-terminal half3,4. The C-terminal half of CYK4 contains a lipid-binding C1 domain that contributes to plasma membrane targeting5 and a GTPase-activating protein (GAP) domain that has an interaction surface for a Rho family GTPase, whose functions have remained unclear 1,3,4,6,7. Here, using the one-cell stage C. elegans embryo as a model, we show that RhoA and the Rho-binding interface of the CYK4 GAP domain drive the recruitment of centralspindlin to the equatorial cortex. By contrast, a point mutant that selectively disrupts GAP activity does not prevent cortical centralspindlin recruitment but instead substantially delays dissipation of centralspindlin from the cortex. These findings suggest that positive feedback, in which centralspindlin recruitment promotes the generation of active RhoA and active RhoA drives centralspindlin recruitment, is central to the rapid assembly of the contractile ring within a narrow time window. They also indicate that the CYK4 GAP catalytic activity contributes to release of centralspindlin from the cortex, potentially to ensure timely dissolution of the contractile ring.

CAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Abstract Background and Aims Others and we have previously demonstrated that cAMP/PKA signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function. Methods The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated over expression of dominant negative (DN) and constitutive active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using cAMP analogue N6-Benzoyl-cAMP (5 mM) and PKI (1 µM), respectively. Pharmacologically, Rac1 was inhibited using NSC 23766 (10 µM) and RhoA was activated using Rho activator I (Cytoskeleton Inc.). In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. Results Pharmacologically, specific activation of PKA induced EC migration (Wound healing assay), tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. These pro-angiogenesis activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA-signalling resulted in an increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and a strong inhibition of RhoA/Rock pathway. Pharmacological activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, over expression of either dominant negative RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis. Conclusion We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/ROCK signalling may offer an important target to intervene therapeutic angiogenesis.

Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation.

Actin and microtubules are essential cytoskeletal components and coordinate their dynamics through multiple coupling and decoupling mechanisms. However, how actin and microtubule dynamics are decoupled remains incompletely understood. Here, we identified TBC1D3C as a new regulator that can decouple actin filament assembly from microtubule disassembly. We showed that TBC1D3C induces the release of GEF-H1 from microtubules into the cytosol without perturbing microtubule arrays, leading to RhoA activation and actin filament assembly. Mechanistically, we found that TBC1D3C directly binds to GEF-H1, disrupting its interaction with the Tctex-DIC-14-3-3 complex and thereby displacing GEF-H1 from microtubules independently of microtubule disassembly. Super-resolution microscopy and live-cell imaging further confirmed that TBC1D3C triggers GEF-H1 release and actin filament assembly while maintaining microtubule integrity. Therefore, our findings demonstrated that TBC1D3C functions as a direct GEF activator and a novel regulator in decoupling actin assembly from microtubule disassembly, providing new insights into cytoskeletal regulation.

A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

Abl kinases regulate FGF signaling independent of Crk phosphorylation to prevent Peters anomaly.

Peters anomaly, the most common cause of congenital corneal opacity, stems from corneal-lenticular adhesion. Despite numerous identified mutations, a cohesive molecular framework of the disease's etiology remains elusive. Here, we identified Abl kinases as pivotal regulators of FGF signaling, as genetic ablation of Abl kinases restores lens induction even in the absence of FGF signaling. Intriguingly, both Abl kinase deficiency and increased FGF-Ras activity result in Peters anomaly independent of ERK signaling, which can be rescued by allelic deletion of Abl substrate, Crk. However, contrary to the prevailing belief that Abl kinases regulate Crk proteins by direct phosphorylation, mutations at Abl kinase phosphorylation sites on Crk and CrkL did not yield any observable effects. Instead, our findings reveal that Abl kinases phosphorylate Ptpn12, which in turn inhibits p130Cas phosphorylation and Crk recruitment, crucial for Rho GTPases activation and cytoskeletal dynamics. Consequently, Abl kinase deficiency reduces actomyosin contractility within the lens vesicle and genetically interacts with RhoA inhibition. Conversely, Rac1 deletion mitigates Peters anomaly in models with aberrant FGF, Abl kinase and RhoA signaling. Our results demonstrate that Abl kinases regulate FGF signaling to balance RhoA and Rac1 activity via the Ptpn12-p130Cas pathway, suggesting that targeting tension-mediated lens vesicle separation could be a therapeutic strategy for Peters anomaly.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Retraction: Chemotherapy Resistance in Diffuse-Type Gastric Adenocarcinoma Is Mediated by RhoA Activation in Cancer Stem-Like Cells.

Retraction: Chemotherapy Resistance in Diffuse-Type Gastric Adenocarcinoma Is Mediated by RhoA Activation in Cancer Stem-Like Cells.

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo, and explored how spermathecal tissue responds to alterations in myosin activity. In animals expressing GFP labeled actin or apical membrane complexes, we severed these structures using femtosecond laser ablation and quantified retractions. RNA interference was used to deplete key contractility regulators. We show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. Our results suggest that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. Additionally, increased tension on the outer basal surface may compress the apical side, leading to lower tensions apically. The three dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

The T-Type Calcium Channel CACNA1H is Required for Smooth Muscle Cytoskeletal Organization During Tracheal Tubulogenesis.

Abnormalities of tracheal smooth muscle (SM) formation are associated with several clinical disorders including tracheal stenosis and tracheomalacia. However, the cellular and molecular mechanisms underlying tracheal SM formation remain poorly understood. Here, it is shown that the T-type calcium channel CACNA1H is a novel regulator of tracheal SM formation and contraction. Cacna1h in an ethylnitrosourea forward genetic screen for regulators of respiratory disease using the mouse as a model is identified. Cacna1h mutants exhibit tracheal stenosis, disorganized SM and compromised tracheal contraction. CACNA1H is essential to maintain actin polymerization, which is required for tracheal SM organization and tube formation. This process appears to be partially mediated through activation of the actin regulator RhoA, as pharmacological increase of RhoA activity ameliorates the Cacna1h-mutant trachea phenotypes. Analysis of human tracheal tissues indicates that a decrease in CACNA1H protein levels is associated with congenital tracheostenosis. These results provide insight into the role for the T-type calcium channel in cytoskeletal organization and SM formation during tracheal tube formation and suggest novel targets for congenital tracheostenosis intervention.

SOXC Enhances NGN2-Mediated Reprogramming of Glioblastoma Cells Into Neuron-Like Cells by Modulating RhoA and RAC1/CDC42 Pathway Activity.

Glioblastoma represents the most frequently diagnosed malignant neoplasm within the central nervous system. Human glioblastoma cells can be phenotypically reprogrammed into neuron-like cells through the forced expression of NEUROG2 and SOXC factors. NEUROG2 serves as a pioneer factor, establishing an initial framework for this transformation. However, the specific role of SOXC factors has not been fully elucidated. In this study, we used ChIP-seq to determine the potential target gene of NGN2. RNA-seq has been used to evaluate the transcriptional change during NGN2-SOX11-mediated neuron reprogramming. Immunofluorescence was used to determine the neuron reprogramming efficacy and cell proliferation ability. ChIP-qPCR, Co-IP, and Western Blot were performed to investigate the mechanism. Our findings reveal that SOXC factors, in contrast to their previously identified function as transcriptional activators, act as transcriptional repressors. They achieve this by recruiting TRIM28 to suppress the expression of ECT2, a RhoGEF. This suppression results in the differential regulation of RhoA, RAC1, and CDC42 activities throughout the reprogramming process. We further establish that small molecules targeting RhoA and its effectors can substitute for SOXC factors in facilitating the neuronal reprogramming of glioblastoma cells. These results underscore the pivotal role of SOXC factors' transcriptional repression and illuminate one of their specific downstream targets.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Tumor-induced endothelial RhoA activation mediates tumor cell transendothelial migration and metastasis.

The endothelial barrier plays an active role in transendothelial tumor cell migration during metastasis, however, the endothelial regulatory elements of this step remain obscure. Here we show that endothelial RhoA activation is a determining factor during this process. Breast tumor cell-induced endothelial RhoA activation is the combined outcome of paracrine IL-8-dependent and cell-to-cell contact β 1 integrin-mediated mechanisms, with elements of this pathway correlating with clinical data. Endothelial-specific RhoA blockade or in vivo deficiency inhibited the transendothelial migration and metastatic potential of human breast tumor and three murine syngeneic tumor cell lines, similar to the pharmacological blockade of the downstream RhoA pathway. These findings highlight endothelial RhoA as a potent, universal target in the tumor microenvironment for anti-metastatic treatment of solid tumors.