RhoA Deficiency Research Articles

Inhibition of RHOA activity preserves the survival and hemostasis function of long-term cold-stored platelets

AbstractPatients with thrombocytopenia require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions, including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis and genetic and pharmacological approaches, we identified that cold-induced activation of RAS homolog family, member A (RHOA) GTPase causes the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage–induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage–induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GPIbα. RHOA inhibition further prevents the metabolic reprogramming of cold storage–induced lesions and allows the maintenance of glycolytic flux and mitochondria-dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room temperature–stored platelets while retaining their hemostatic activity in vivo, as assessed by bleeding time correction in aspirin-treated mice. Our studies provide a mechanism-based translational approach to prevent cold storage–induced damage, which is useful for human platelet transfusion in patients with thrombocytopenia.

RHOA deficiency drives decreased CD19 expression and immune dysregulation in CAR‐T resistant diffuse large B‐cell lymphoma

Background: CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new options in relapsed or refractory diffuse large B-cell lymphoma (DLBCL), but over half of patients still progress. Recent whole-genome sequencing data published by our lab revealed that deletion of RHOA (3p21.31) is strongly associated with poor response (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-19 resistant tumors. Methods: RHOA loss-of-function (LoF) lymphoma cell lines were generated using shRNA and CRISPR approaches. Diminished RHOA activity was confirmed via active RHO pulldowns. RNA-sequencing in conjunction with western blotting and flow cytometry was conducted to uncover deregulated pathways in RHOA-deficient lymphoma. Cell proliferation, migration, and viability were assessed to phenotype RHOA LoF and uncover signaling pathways activated in RHOA-deficient lymphoma. CD19-directed murine and human CAR-T cells were co-cultured with corresponding RHOA LoF systems to assess CAR-T engagement and killing. Results: RNA-seq analysis of RHOA-deficient cell lines revealed enrichment of gene sets associated with cell-cycle progression and interferon signaling pathways, and cell proliferation and chemotaxis assays both demonstrated increased activation. Western blotting revealed upregulated pAKT, but strikingly RHOA LoF systems showed enhanced sensitivity to PI3K, AKT, and mTOR inhibition. We assessed if RHOA LoF associates with previously described decreased CD19 driving CAR-19 resistance and found consistent downregulation by flow cytometry in both A20 shRhoa (p = 0.0002) murine lymphomas and RIVA human cell line CRISPR het RHOA knockouts (p = 0.0031), confirmed by western blotting in both systems. RNA-seq analysis of 74 DLBCL patients (PCAWG data) revealed a strong positive correlation between expression CD19 and RHOA (p < 0.001, R2 = 0.236). Impedance-based serial cell viability assessments and flow cytometry cytotoxicity assays with CAR-19 and RHOA LoF cells revealed a statistically significant decrease in CAR-19 killing of RHOA-deficient lymphomas (p = 0.0005 and p < 0.0001). Conclusion: RHOA-deficient lymphoma relies on PI3K/AKT/mTOR signaling for proliferation that works in parallel with decreased CD19 to decrease CAR-19 killing. Upregulation of interferon signaling pathways gives evidence of increased immunoinhibitory ligands that could promote exhausted CAR19 products. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Immunotherapy, Microenvironment No conflicts of interests pertinent to the abstract.

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling

BackgroundThe cytoskeletal architecture of osteoclasts (OCs) and bone resorption activity must be appropriately controlled for proper bone remodeling, which is associated with osteoporosis. The RhoA protein of GTPase plays a regulatory role in cytoskeletal components and contributes to osteoclast adhesion, podosome positioning, and differentiation. Although osteoclast investigations have traditionally been performed by in vitro analysis, however, the results have been inconsistent, and the significance of RhoA in bone physiology and pathology is still unknown.MethodsWe generated RhoA knockout mice by specifically deleting RhoA in the osteoclast lineage to understand more about RhoA’s involvement in bone remodeling. The function of RhoA in osteoclast differentiation and bone resorption and the mechanisms were assessed using bone marrow macrophages (BMMs) in vitro. The ovariectomized (OVX) mouse model was adopted to examine the pathological effect of RhoA in bone loss.ResultsConditional deletion of RhoA in the osteoclast lineage causes a severe osteopetrosis phenotype, which is attributable to a bone resorption suppression. Further mechanistic studies suggest that RhoA deficiency suppresses Akt-mTOR-NFATc1 signaling during osteoclast differentiation. Additionally, RhoA activation is consistently related to the significant enhancement the osteoclast activity, which culminates in the development of an osteoporotic bone phenotype. Furthermore, in mice, the absence of RhoA in osteoclast precursors prevented occurring OVX-induced bone loss.ConclusionRhoA promoted osteoclast development via the Akt-mTOR-NFATc1 signaling pathway, resulting a osteoporosis phenotype, and that manipulating RhoA activity might be a therapeutic strategy for osteoporotic bone loss.

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity.

Statins are widely prescribed cholesterol-lowering drugs but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanisms underlying the diabetogenic effect of statins are still not fully understood. The effects of geranylgeranyl transferase I and II (GGTase I and II) inhibition on insulin-stimulated glucose uptake and GLUT4 translocation, and the dependence of these effects on insulin signalling were investigated in skeletal muscle cells. The protective effects of geranylgeranyl pyrophosphate (GGPP) and its precursor geranylgeraniol (GGOH) on simvastatin-induced insulin resistance were evaluated in vitro and in vivo. The effect of GGTase II inhibition in skeletal muscle on insulin sensitivity in vivo was confirmed by adeno-associated virus serotype 9 (AAV9)-mediated knockdown of the specific subunit of GGTase II, RABGGTA. The regulatory mechanisms of GGTase I on insulin signalling and GGTase II on insulin-stimulated GLUT4 translocation were investigated by knockdown of RhoA, TAZ, IRS1, geranylgeranylation site mutation of RhoA, RAB8A, and RAB13. Both inhibition of GGTase I and II mimicked simvastatin-induced insulin resistance in skeletal muscle cells. GGPP and GGOH were able to prevent simvastatin-induced skeletal muscle insulin resistance in vitro and in vivo. GGTase I inhibition suppressed the phosphorylation of AKT (Ser473) (-51.3%, P<0.01), while GGTase II inhibition had no effect on it. AAV9-mediated knockdown of RABGGTA in skeletal muscle impaired glucose disposal without disrupting insulin signalling in vivo (-46.2% for gastrocnemius glucose uptake, P<0.001; -52.5% for tibialis anterior glucose uptake, P<0.001; -17.8% for soleus glucose uptake, P<0.05; -31.4% for extensor digitorum longus glucose uptake, P<0.01). Inhibition of RhoA, TAZ, IRS1, or geranylgeranylation deficiency of RhoA attenuated the beneficial effect of GGPP on insulin signalling in skeletal muscle cells. Geranylgeranylation deficiency of RAB8A inhibited insulin-stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells (-42.8% for GLUT4 translocation, P<0.01; -50.6% for glucose uptake, P<0.001). Geranylgeranyl pyrophosphate regulates glucose uptake via GGTase I-mediated insulin signalling-dependent way and GGTase II-mediated insulin signalling-independent way in skeletal muscle. Supplementation of GGPP/GGOH could be a potential therapeutic strategy for statin-induced insulin resistance.

Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice

AbstractMegakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro–generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.

Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the β1 integrin-RhoA-formin axis.

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

Endothelial Small GTPase RhoA: A Potential Target for Anti‐angiogenic Therapy

ObjectiveTumor vessels display abnormal structure and function due to imbalanced angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best known pro‐angiogenic factors in this process. The goal of antiangiogenic therapy is to inhibit tumor growth by oxygen and nutrient deprivation. Current antiangiogenic therapies target VEGF/VEGFR signaling pathways. However development of resistance due to upregulation of bFGF remains a serious disadvantage of current approaches, leading to efforts of simultaneous targeting of both VEGF and FGF pathways. A small GTPase RhoA has been reported to regulate VEGF‐induced angiogenesis. In the present study we investigate the role of endothelial RhoA in bFGF‐induced angiogenesis to identify whether RhoA can be considered a target for both VEGF‐ and FGF‐induced angiogenesis.MethodsWe performed RhoA pulldown assays to identify RhoA activation downstream of bFGF treatment. We used Boyden chamber cell migration assay, 2D tube formation assay using matrigel and 3D sprouting assay with collagen to assess angiogenic potential. Endothelial RhoA was knocked down by siRNAs or blocked by pharmacological inhibitors. Biochemical assays were performed to identify the molecular mechanism of bFGF‐induced RhoA activation. In vivo, endothelial‐specific inducible RhoA‐deficient mice with a fluorescent reporter were used in a newly modified matrigel plug angiogenesis model allowing identification of endothelial RhoA excision efficiency.ResultsbFGF treatment activates RhoA in human umbilical vein endothelial Cells (HUVEC). RhoA inhibition with C3 toxin or RhoA knockdown abrogated bFGF‐induced endothelial cell migration and tube formation in vitro. In vivo, endothelial RhoA deficiency blocked bFGF‐induced angiogenesis in matrigel plug assay. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF‐induced RhoA activation and angiogenesis. RhoA inhibition partially blocked bFGF‐induced JNK phosphorylation but did not affect ERK and p38 activation. Ongoing experiments are aiming to delineate the precise role of endothelial RhoA in the FGF downstream pathway.ConclusionCollectively, our data suggest that RhoA pathway participates in bFGF‐induced angiogenesis and can be a potential target for anti angiogenic therapy.

Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis

Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.

Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

Asthma is a heterogeneous chronic airway inflammation in which Th2 and Th17 cells are key players in its pathogenesis. We have reported that RhoA of Rho GTPases orchestrated glycolysis for Th2 cell differentiation and allergic airway inflammation by the use of a conditional RhoA-deficient mouse line. However, the role of RhoA in Th17 cells remains to be elucidated. In this study, we investigated the effects of RhoA deficiency on Th17 cells in the context of ex vivo cell culture systems and an in vivo house dust mites (HDM)-induced allergic airway inflammation. We found that RhoA deficiency inhibited Th17 differentiation and effector cytokine secretion, which was associated with the downregulations of Stat3 and Rorγt, key Th17 transcription factors. Furthermore, loss of RhoA markedly suppressed Th17 and neutrophil-involved airway inflammation induced by HDM in mice. The infiltrating inflammatory cells in the lungs and bronchoalveolar lavage (BAL) fluids were dramatically reduced in conditional RhoA-deficient mice. Th17 as well as Th2 effector cytokines were suppressed in the airways at both protein and mRNA levels. Interestingly, Y16, a specific RhoA inhibitor, was able to recapitulate the most phenotypes of RhoA genetic deletion in Th17 differentiation and allergic airway inflammation. Our data demonstrate that RhoA is a key regulator of Th17 cell differentiation and function. RhoA might serve as a potential novel therapeutic target for asthma and other inflammatory disorders.

Endothelial RhoA Regulates Breast Cancer Metastasis

Metastasis is the cause of more than 90% of cancer‐related deaths. An important step in metastatic process is the migration of the disseminating cancer cells through the endothelial monolayer during entrance or exit from the vasculature. Activation of endothelial RhoA‐ROCK pathway has been implicated in the regulation of vascular permeability in various pathophysiological conditions. In the present study, we have investigated the role of this pathway in trans‐endothelial migration and metastasis of breast cancer cells. RhoA activation was identified by RhoA pull‐down experiments of murine and human breast cancer cell supernatant on primary endothelial cells. Cancer cell trans‐endothelial migration efficiency was explored through an in vitro transwell‐based two‐cell co‐culture model. RhoA signaling pathway was blocked by both pharmacological inhibition and knockdown experiments. In vivo metastatic potential was identified by experimental metastasis models of murine and human breast cancer cell lines using endothelial‐specific RhoA‐deficient mice or clinically relevant ROCK inhibitors. We identified that the diverse metastatic potential of a panel of human breast cancer cell lines correlated with the RhoA activation efficiency of their supernatant on primary endothelial cells. Secretome analysis revealed that IL‐8 (over)expression was relevant to breast cancer cell aggressiveness and its functionality was tested with gain‐and loss‐of‐function experiments. In vivo, experimental metastasis experiments with the syngeneic breast cancer cell line E0771 showed that endothelial RhoA deficiency decreased the metastatic potential. Similar inhibition was obtained with treatment with the clinically‐relevant ROCK inhibitor, Fasudil, in experimental metastasis experiments of murine and human cell lines using immune‐competent and immune‐deficient mice respectively. Collectively, our in vitro and in vivo findings demonstrate that the endothelial RhoA‐ROCK pathway affects breast cancer trans‐endothelial migration and metastasis and propose endothelial RhoA inhibition as a novel therapy for breast cancer metastases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RHOA GTPase Controls YAP-Mediated EREG Signaling in Small Intestinal Stem Cell Maintenance.

SummaryRHOA, a founding member of the Rho GTPase family, is critical for actomyosin dynamics, polarity, and morphogenesis in response to developmental cues, mechanical stress, and inflammation. In murine small intestinal epithelium, inducible RHOA deletion causes a loss of epithelial polarity, with disrupted villi and crypt organization. In the intestinal crypts, RHOA deficiency results in reduced cell proliferation, increased apoptosis, and a loss of intestinal stem cells (ISCs) that mimic effects of radiation damage. Mechanistically, RHOA loss reduces YAP signaling of the Hippo pathway and affects YAP effector epiregulin (EREG) expression in the crypts. Expression of an active YAP (S112A) mutant rescues ISC marker expression, ISC regeneration, and ISC-associated Wnt signaling, but not defective epithelial polarity, in RhoA knockout mice, implicating YAP in RHOA-regulated ISC function. EREG treatment or active β-catenin Catnblox(ex3) mutant expression rescues the RhoA KO ISC phenotypes. Thus, RHOA controls YAP-EREG signaling to regulate intestinal homeostasis and ISC regeneration.

Raf-1/CK2 and RhoA/ROCK signaling promote TNF-α-mediated endothelial apoptosis via regulating vimentin cytoskeleton.

Both RhoA/ROCK and Raf-1/CK2 pathway play essential roles in cell proliferation, apoptosis, differentiation, and multiple other common cellular functions. We previously reported that vimentin is responsible for TNF-α-induced cell apoptosis. Herein, we investigated the regulation of RhoA/ROCK and Raf-1/CK2 signaling on vimentin filaments and endothelial apoptosis mediated by TNF-α. Treatment with TNF-α significantly induced the activation of RhoA and ROCK, and the expression of ROCK1. RhoA deficiency could obviously inhibit ROCK activation and ROCK1 expression induced by TNF-α. Both RhoA deficiency and ROCK activity inhibition (Y-27632) greatly inhibited endothelial apoptosis and preserved cell viability in TNF-α-induced human umbilical vein endothelial cells (HUVECs). Also vimentin phosphorylation and the remodeling of vimentin or phospho-vimentin induced by TNF-α were obviously attenuated by RhoA suppression and ROCK inhibition. TNF-α-mediated vimentin cleavage was significantly inhibited by RhoA suppression and ROCK inhibition through decreasing the activation of caspase3 and 8. Furthermore, TNF-α treatment greatly enhanced the activation of Raf-1. Suppression of Raf-1 or CK2 by its inhibitor (GW5074 or TBB) blocked vimentin phosphorylation, remodeling and endothelial apoptosis, and preserved cell viability in TNF-α-induced HUVECs. However, Raf-1 inhibition showed no significant effect on TNF-α-induced ROCK expression and activation, suggesting that the regulation of Raf-1/CK2 signaling on vimentin was independent of ROCK. Taken together, these results indicate that both RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-α-mediated endothelial cytotoxicity via regulating vimentin cytoskeleton.

Reciprocal regulation of Th17 and Treg cell differentiation by RhoA

Abstract Th17 cells are important for protective immunity as well as tissue inflammation and autoimmune disorders. Treg cells, including induced Treg (iTreg) and natural Treg (nTreg), play a critical role in immune tolerance. RhoA of the Rho small GTPase family is an intracellular signal transducer that cycles between an inactive GDP-bound form and an active GTP-bound form. We have previously reported that RhoA plays an important role in Th2 cell differentiation by regulating glycolysis. In this study, we further examined the role of RhoA in Th17 and Treg cell differentiation. Genetic deletion of RhoA in T cells or pharmacologic inhibition of RhoA by a RhoA-specific inhibitor Y16 led to reduced Th17 differentiation in vitro as evidenced by reduction of Th17 effector cytokines, transcriptional downregulation of RORγT and reduced phosphorylation of Stat3. Accordingly, RhoA deficiency ameliorated house dust mites (HDM)-induced allergic airway inflammation and dextran sodium sulphate (DSS)-induced colitis in vivo where Th17 cells plays an important role. Inhibition of ROCK, an immediate downstream effector of RhoA, recapitulated the effect of RhoA deficiency or inhibition on Th17 differentiation, suggesting that RhoA regulates Th17 cell differentiation through ROCK. On the other hand, ablation of RhoA had no effect on iTreg cell differentiation. However, inhibition of ROCK enhanced iTreg differentiation, suggesting that ROCK has RhoA-independent role in iTreg differentiation. In contrast to its in-effect on iTreg differentiation, RhoA deficiency increased nTreg differentiation. Thus, our data have provided new insights into Th17 and Treg differentiation and identified RhoA as a potential therapeutic target for tissue inflammation.

RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal.

BackgroundPodocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown.MethodsCulture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes.ResultsWe found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis.ConclusionRhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases.

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation

AbstractRhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.5 (E16.5). The primitive red blood cells were enlarged, poikilocytic, and frequently multinucleated, but were able to sustain life despite experiencing cytokinesis failure. In contrast, definitive erythropoiesis failed and the mice died by E16.5, with profound reduction of maturing erythroblast populations within the fetal liver. RhoA was required to activate myosin-regulatory light chain and localized at the site of the midbody formation in dividing wild-type erythroblasts. Cytokinesis failure caused by RhoA deficiency resulted in p53 activation and p21-transcriptional upregulation with associated cell-cycle arrest, increased DNA damage, and cell death. Our findings demonstrate the role of RhoA as a critical regulator for efficient erythroblast proliferation and the p53 pathway as a powerful quality control mechanism in erythropoiesis.

RhoA orchestrates glycolysis for TH2 cell differentiation and allergic airway inflammation

Mitochondrial metabolism is known to be important for T-cell activation. However, its involvement in effector T-cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T-cell activation and effector cell differentiation and function remains largely unknown. We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for TH2 cell differentiation and TH2-mediated allergic airway inflammation. Conditional RhoA-deficient mice were generated by crossing RhoA(flox/flox) mice with CD2-Cre transgenic mice. Effects of RhoA on TH2 differentiation were evaluated based on invitro TH2-polarized culture conditions and invivo in ovalbumin-induced allergic airway inflammation. Cytokine levels were measured by using intracellular staining and ELISA. T-cell metabolism was measured by using the Seahorse XF24 Analyzer and flow cytometry. Disruption of RhoA inhibited T-cell activation and TH2 differentiation invitro and prevented the development of allergic airway inflammation invivo, with no effect on TH1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways, such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to TH2 cell differentiation and allergic airway inflammation through regulating IL-4 receptor mRNA expression and TH2-specific signaling events. Finally, inhibition of Rho-associated protein kinase, an immediate downstream effector of RhoA, blocked TH2 differentiation and allergic airway inflammation. RhoA is a key component of the signaling cascades leading to TH2 differentiation and allergic airway inflammation at least in part through control of T-cell metabolism and the Rho-associated protein kinase pathway.

RhoA Gtpase Regulates Erythroblast Cytokinesis and Enucleation By Dynamic Coordination Of The Microtubule-Actomyosin Machineries For Successful Abscission

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012).To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

RhoA GTPase controls cytokinesis and programmed necrosis of hematopoietic progenitors

Hematopoietic progenitor cells (HPCs) are central to hematopoiesis as they provide large numbers of lineage-defined blood cells necessary to sustain blood homeostasis. They are one of the most actively cycling somatic cells, and their precise control is critical for hematopoietic homeostasis. The small GTPase RhoA is an intracellular molecular switch that integrates cytokine, chemokine, and adhesion signals to coordinate multiple context-dependent cellular processes. By using a RhoA conditional knockout mouse model, we show that RhoA deficiency causes a multilineage hematopoietic failure that is associated with defective multipotent HPCs. Interestingly, RhoA(-/-) hematopoietic stem cells retained long-term engraftment potential but failed to produce multipotent HPCs and lineage-defined blood cells. This multilineage hematopoietic failure was rescued by reconstituting wild-type RhoA into the RhoA(-/-) Lin(-)Sca-1(+)c-Kit(+) compartment. Mechanistically, RhoA regulates actomyosin signaling, cytokinesis, and programmed necrosis of the HPCs, and loss of RhoA results in a cytokinesis failure of HPCs manifested by an accumulation of multinucleated cells caused by failed abscission of the cleavage furrow after telophase. Concomitantly, the HPCs show a drastically increased death associated with increased TNF-RIP-mediated necrosis. These results show that RhoA is a critical and specific regulator of multipotent HPCs during cytokinesis and thus essential for multilineage hematopoiesis.

Erythroid Specific RhoA Deficiency Causes Dysplastic and Inefficient Fetal Erythropoiesis with Lethal Embryonic Anemia Due to Defects in Erythroblast Cytokinesis and Enucleation

Abstract 82RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis (Schwartz, J Cell Sci 2004). We recently demonstrated evidence that enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. We showed that RhoA-related Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation (Konstantinidis et al, Blood 2012).Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesized that RhoA dynamically controls erythroblast enucleation by molecular pathways analogous to the ones involved in cytokinesis. To define the mechanistic contribution of RhoA signaling in erythropoiesis and enucleation, we generated mice with erythroid specific deletion (EpoRGFPcre/+ driven) of RhoA. Erythroid-specific deletion of RhoA caused severe anemia, which was fatal in utero by E14.5. These embryos as well as their yolk sacs had a pallid, anemic appearance and their peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers at E12.5 had approximately 33% of the cellularity of WT littermates (n=9, p<0.001). Analysis of definitive erythropoiesis in the RhoAΔ/Δ fetal liver showed that despite decreased cellularity, the erythroid progenitors BFU-E and CFU-E were not different in total number per fetal liver. In contrast, the erythroblast populations, as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoA-deficient fetal liver erythroblasts expressed significantly higher levels of the α4 and α5 integrin subunits, suggesting a compensatory increase of the adhesion receptors due to impaired downstream signaling. We further examined the erythroblastic islands in RhoAΔ/Δ fetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were more loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, that has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010). Upon evaluation of the RhoA protein level and phosphorylated myosin regulatory light chain (pMRLC) by immunoblotting of RhoAΔ/Δ fetal liver cells, pMRLC was found significantly decreased, indicating that one of the signaling pathways regulated by RhoA in erythropoiesis involves phosphorylation of MRLC. Moreover, treatment of wild type erythroblasts in in vitro erythropoiesis assays with the pharmacological inhibitor Y27632 of Rho-associated protein kinase (ROCK), a downstream target of RhoA participating in myosin regulation, resulted in a decrease of enucleation by up to 38% (n=3, p<0.05).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation via regulation of the actomyosin cytoskeleton by ROCK-mediated phosphorylation of MRLC. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Further exploration of the role of RhoA in erythropoiesis has the potential to reveal targets for therapeutic interventions for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

Podocyte-specific loss of Cdc42 leads to congenital nephropathy.

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.