GEFs on the RhoAd to a colossal nucleus

Cytokinesis in normal cell division requires RhoA-regulated actomyosin contraction of the cleavage furrow; this process is aborted in megakaryocyte endomitosis, leading to polyploidy. In this issue of Developmental Cell, Gao et al. (2012) trace the basis of endomitosis to sequential downregulation of guanine nucleotide exchange factors GEF-H1 and ECT2.     

RhoA and Rho kinase mediate cyclosporine A and sirolimus-induced barrier tightening in renal proximal tubular cells

The regulation and maintenance of the paracellular transport in renal tubular epithelia is vital for kidney functions. Combination of the immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) exerts powerful immunosuppression, but also causes nephrotoxicity. We have previously shown that CsA and SRL elevate transepithelial resistance (TER) in kidney tubular cells partly through MEK/ERK1/2. In this work we examined the hypothesis that the RhoA pathway may also be mediating effects of CsA and SRL. We show that CsA and the CsA/SRL combination activated RhoA, induced cofilin phosphorylation and promoted stress fiber generation. The Rho kinase (ROK) inhibitor, Y27632, prevented CsA and CsA/SRL-induced cofilin phosphorylation and actin remodelling, reduced the TER increase and prevented the rise in claudin-7 levels caused by the drugs. Expression of the exchange factor GEF-H1/lfc was elevated in cells treated with CsA and CsA/SRL. GEF-H1 silencing inhibited RhoA activation by ≈50%, and potently reduced cofilin phosphorylation and stress fiber formation induced by CsA and CsA/SRL. However, GEF-H1 downregulation did not prevent the TER change. Thus the Rho/Rho kinase pathway was involved in mediating CsA and CsA/SRL-induced cytoskeleton rearrangement and TER changes via claudin-7 expression. Our data however point to differential regulation of Rho activation involved in central cytoskeleton remodelling, that is GEF-H1-dependent and junctional permeability that does not require GEF-H1.     

mTOR／4E—BP1参与诺考达唑诱导的Dami细胞多倍体化调控

目的：多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程（如恶性肿瘤）的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR／S6K1参与多倍体细胞周期的调控。本课题主要研究roTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法：诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR／4E—BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E—BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果：诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E—BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2／M期。结论：mTOR／4E-BP1通路参与多倍体细胞周期的调控。     

The role of 2n gametes and endosperm balance number in the origin and evolution of polyploids in the tuber-bearing Solanums

Polyploidization has played a major role in the origin and evolution of polyploid species. In this article we outline the unique characteristics of 2n gametes and implications of their participation in the evolution of polyploid Solanum species. The genetic consequences of 2n gametes indicate that sexual polyploidization results in greater variability, fitness, and heterozygosity than does somatic doubling. Further, the mechanisms of 2n gamete formation and the frequency of 2n gamete-forming genes in present polyploids and their ancestral species provide additional evidence of their involvement. Equally important is the endosperm, via the endosperm balance number (EBN) incompatibility system, in complementing the role of 2n gametes. In fact, the EBN system acts as a screen for either 1n or 2n gametes, depending on the EBN and chromosome numbers of parental species. EBN in combination with 2n gametes maintains the ploidy integrity of diploid ancestral species, while providing the flexibility for either unilateral or bilateral sexual polyploidization.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.     

Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition

The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.     

Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.     

Heparanase-induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

Heparanase is the only mammalian endoglycosidase which has been widely implicated in cancer because of its capability to degrade heparan sulfate chains of heparan sulfate proteoglycans (HSPG). Specifically, the cell surface HSPG syndecan-1 and -4 (SDC1 and SDC4) are modulators of growth factor action, and SDC4 is implicated in cell adhesion as a key member of focal adhesion complexes. We hypothesized that extracellular heparanase modulates brain metastatic breast cancer (BMBC) cell invasiveness by affecting cytoskeletal dynamics, SDC4 carboxy-terminal-associated proteins, and downstream targets. We used two independently derived human BMBC cell systems (MB-231BR and MB-231BR3), which possess distinct cellular morphologies and properties. Highly aggressive spindle-shaped 231BR3 cells changed to a round cell morphology associated with expression of the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1). We showed that GEF-H1 is a new component of the SDC4 signaling complex in BMBC cells. Treatment with heparanase resulted in regulation of the SDC4/protein kinase C α axis while maintaining a constitutive GEF-H1 level. Third, GEF-H1 knockdown followed by cell exposure to heparanase caused a significant regulation of activities of Rac1 and RhoA, which are GEF-H1 targets and fundamental effectors in cell plasticity control. Fourth, L-heparanase augmented expression of β1 integrin in BMBC cells and of vascular cell adhesion molecule 1 (VCAM1; the major β1 integrin receptor) in human brain microvascular endothelial cells. Finally, using a newly developed blood-brain barrier in vitro model, we show that BMBC cell transmigration was significantly reduced in GEF-H1 knockdown cells. These findings implicate heparanase in mechanisms of cytoskeletal dynamics and in the cross-talk between tumor cells and vascular brain endothelium. They are of relevance because they elucidate molecular events in the initial steps leading to BMBC onset and capturing distinct roles of latent and active heparanase in the brain microenvironment.     

Dynamic regulation of GEF-H1 localization at microtubules by Par1b/MARK2

Par1b/MARK2 is a serine/threonine kinase that plays key roles in the development of cell polarity, but its precise mechanism of action remains unknown. Here we report that GEF-H1, a guanine nucleotide exchange factor for Rho-family small GTPases, is a novel substrate for Par1b. GEF-H1 directly associates with microtubules via its N-terminal C1 domain, which is known to regulate the activity of GEF-H1. Ectopically expressed GEF-H1 markedly promotes stabilization of microtubules, resulting in acetylation of microtubules. We find that Par1b phosphorylates GEF-H1 at three serine residues conserved in vertebrates and releases GEF-H1 from microtubules, which abrogates stabilization and acetylation of microtubules induced by GEF-H1 overexpression. The alanine mutant for the three phosphorylation sites (3SA) of GEF-H1 strongly induces stabilization and acetylation of microtubules, which was resistant to Par1b. Time-lapse imaging analyses reveal that GFP-fused GEF-H1 dynamically moved on microtubules from one protrusion to another, whereas the 3SA mutant was static. These data suggest that Par1b-phosphorylation regulates turnover of GEF-H1 localization by regulating its interaction with microtubules, which may contribute to cell polarization.     

Bone marrow niche-inspired,multiphase expansion of megakaryocytic progenitors with high polyploidization potential

Background aimsMegakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO₂ increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.MethodsCD34⁺ HSPCs were cultured at pH 7.2 and 5% O₂ with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O₂ to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.ResultsUsing Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34⁺ CD41⁺ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO₂. Although fewer progenitors were present, shifting to 20% O₂/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.ConclusionsWe obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.     

Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.     

植物多倍体化中基因组和基因表达的变化

多倍体化在植物进化的历史过程中频繁发生, 对新物种的形成产生了很大影响。伴随着多倍体化, 植物在基因组和基因表达上发生了复杂的变化, 包括染色体数目变化、染色体重组、基因沉默、基因的非加性表达和表观遗传等变化。该文对多倍体化引起的这些变化及其相应的机理进行了综述, 以期为了解多倍体化中植物新表型的产生机理和在进化中的意义提供参考。     

Contribution of guanine exchange factor H1 in phorbol ester-induced apoptosis

Phorbol-12-myristate-13-acetate (PMA) treatment induces erythroblastoma D2 cells kept in suspension to undergo RhoA-dependent contraction and to become proapoptotic, while attached cells are induced to differentiate accompanied by the reduction of RhoA activity. In this study, we found that guanine exchange factor H1 (GEF-H1) is highly expressed in D2 cells. Depletion of GEF-H1 expression in D2 cells decreased RhoA activity and prevented PMA-induced contraction and apoptosis. Upon PMA stimulation, GEF-H1 became associated with microtubules in cells that were induced to differentiate. As a contrast, in the proapoptotic population of cells GEF-H1 stayed in the cytoplasm without showing PMA-responsive microtubule translocation. Given that GEF-H1 is inactivated when associated with microtubules and its release into cytosol due to depolymerization of microtubules activates RhoA, our results demonstrated that nonmicrotubule-associated GEF-H1 in D2 cells contributes to the sustained activation of RhoA/ROCK signaling in suspension cells, making cells susceptible to PMA-induced apoptosis.     

A Highlights from MBoC Selection: The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.     

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.     

Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis

The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.     

Extracellular Signal-Regulated Kinase Regulates RhoA Activation and Tumor Cell Plasticity by Inhibiting Guanine Exchange Factor H1 Activity

In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.     

Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N-terminal cell cycle regulator-related domains

The ECT2 protooncogene plays a critical role in cytokinesis, and its C-terminal half encodes a Dbl homology-pleckstrin homology module, which catalyzes guanine nucleotide exchange on the Rho family of small GTPases. The N-terminal half of ECT2 (ECT2-N) contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6, and fission yeast Cut5. The Cut5-related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins. ECT2 is ubiquitously expressed in various tissues and cell lines, but elevated levels of ECT2 expression were found in various tumor cell lines and rapidly developing tissues in mouse embryos. Consistent with these findings, induction of ECT2 expression was observed upon stimulation by serum or various growth factors. In contrast to other oncogenes whose expression is induced early in G1, ECT2 expression was induced later, coinciding with the initiation of DNA synthesis. To test the role of the cell cycle regulator/checkpoint control protein-related domains of ECT2 in cytokinesis, we expressed various ECT2 derivatives in U2OS cells, and analyzed their DNA content by flow cytometry. Expression of the N-terminal half of ECT2, which lacks the catalytic domain, generated cells with more than 4N DNA content, suggesting that cytokinesis was inhibited in these cells. Interestingly, ECT2-N lacking the nuclear localization signals inhibited cytokinesis more strongly than the derivatives containing these signals. Mutational analyses revealed that the XRCC1, CLB6, and BRCT domains in ECT2-N are all essential for the cytokinesis inhibition by ECT2-N. These results suggest that the XRCC1, CLB6, and BRCT domains of ECT2 play a critical role in regulating cytokinesis.