scholarly journals The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling

2015 ◽  
Vol 211 (6) ◽  
pp. 1177-1192 ◽  
Author(s):  
Costanza Giampietro ◽  
Andrea Disanza ◽  
Luca Bravi ◽  
Miriam Barrios-Rodiles ◽  
Monica Corada ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Intracellular Signals ◽  
Transcriptional Cofactors

Vascular endothelial (VE)–cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14–3-3–YAP associates with the VE–cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

scholarly journals Modulating PKCα Activity to Target Wnt/β-Catenin Signaling in Colon Cancer

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 693 ◽  
Author(s):  
Sébastien Dupasquier ◽  
Philippe Blache ◽  
Laurence Picque Lasorsa ◽  
Han Zhao ◽  
Jean-Daniel Abraham ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Tumor Development ◽  
Orphan Receptor ◽  
Nuclear Accumulation ◽  
Kinase C ◽  
Extracellular Signal

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/β-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/β-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-β/axin destruction complex of β-catenin. This results in sustained nuclear accumulation of β-catenin, followed by β-catenin-dependent co-transcriptional activation of Wnt/β-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling β-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits β-catenin co-transcriptional activity. PKCα also phosphorylates β-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

scholarly journals New insights into cadherin function in epidermal sheet formation and maintenance of tissue integrity

2008 ◽  
Vol 105 (40) ◽  
pp. 15405-15410 ◽  
Author(s):  
Christopher L. Tinkle ◽  
H. Amalia Pasolli ◽  
Nicole Stokes ◽  
Elaine Fuchs
Keyword(s):  
Adherens Junction ◽  
Actin Dynamics ◽  
Loss Of Function ◽  
Cortical Actin ◽  
Tissue Integrity ◽  
Gene Ablation ◽  
Rnai Technology ◽  
Physiological Relevance

Co-expression and gene linkage have hampered elucidating the physiological relevance of cadherins in mammalian tissues. Here, we combine conditional gene ablation and transgenic RNA interference to uncover new roles for E- and P-cadherins in epidermal sheet formation in vitro and maintenance of epidermal integrity in vivo. By devising skin-specific RNAi technology, we demonstrate that cadherin inhibition in vivo impairs junction formation and intercellular adhesion and increases apoptosis. These defects compromise epidermal barrier function and tissue integrity. In vitro, with only E-cadherin missing, epidermal sheet formation is delayed, but when both cadherins are suppressed, defects extend to adherens junctions, desmosomes, tight junctions and cortical actin dynamics. Using different rescue strategies, we show that cadherin level rather than subtype is critical. Finally, by comparing conditional loss-of-function studies of epidermal catenins and cadherins, we dissect cadherin-dependent and independent roles of adherens junction components in tissue physiology.

scholarly journals Caveolin-1-Mediated Tumor Suppression Is Linked to Reduced HIF1α S-Nitrosylation and Transcriptional Activity in Hypoxia

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2349
Author(s):  
Carlos Sanhueza ◽  
Jimena Castillo Bennett ◽  
Manuel Valenzuela-Valderrama ◽  
Pamela Contreras ◽  
Lorena Lobos-González ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Tumor Suppression ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Caveolin 1 ◽  
Oxide Synthase ◽  
E Cadherin

Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.

scholarly journals Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Eric S. Sorensen ◽  
Amanda B. Macedo ◽  
Rachel S. Resop ◽  
J. Natalie Howard ◽  
Racheal Nell ◽  
...  
Keyword(s):  
Biological Activity ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Cell Model ◽  
Systematic Analysis ◽  
Immune Effector ◽  
Relationship Analysis ◽  
Latent Hiv

ABSTRACT “Shock and kill” therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo. A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Regulation of the chondrocyte phenotype by β-catenin

Development ◽  
2002 ◽  
Vol 129 (23) ◽  
pp. 5541-5550
Author(s):  
Je-Hwang Ryu ◽  
Song-Ja Kim ◽  
Seon-Hee Kim ◽  
Chun-Do Oh ◽  
Sang-Gu Hwang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Monolayer Culture ◽  
Nuclear Translocation ◽  
Articular Chondrocytes ◽  
Glycogen Synthase ◽  
Ectopic Expression ◽  
Activator Protein 1 ◽  
Chondrocyte Phenotype

β-Catenin regulates important biological processes, including embryonic development and tumorigenesis. We have investigated the role ofβ-catenin in the regulation of the chondrocyte phenotype. Expression ofβ-catenin was high in prechondrogenic mesenchymal cells, but significantly decreased in differentiated chondrocytes both in vivo and in vitro. Accumulation of β-catenin by the inhibition of glycogen synthase kinase-3β with LiCl inhibited chondrogenesis by stabilizing cell-cell adhesion. Conversely, the low level of β-catenin in differentiated articular chondrocytes was increased by post-translational stabilization during phenotypic loss caused by a serial monolayer culture or exposure to retinoic acid or interleukin-1β. Ectopic expression of β-catenin or inhibition of β-catenin degradation with LiCl or proteasome inhibitor caused de-differentiation of chondrocytes. Transcriptional activation ofβ-catenin by its nuclear translocation was sufficient to cause phenotypic loss of differentiated chondrocytes. Expression pattern of Jun, a known target gene of β-catenin, is essentially the same as that of β-catenin both in vivo and in vitro suggesting that Jun and possibly activator protein 1 is involved in the β-catenin regulation of the chondrocyte phenotype.

scholarly journals In Vivo Importance of Actin Nucleotide Exchange Catalyzed by Profilin

2000 ◽  
Vol 150 (4) ◽  
pp. 895-904 ◽  
Author(s):  
Amy K. Wolven ◽  
Lisa D. Belmont ◽  
Nicole M. Mahoney ◽  
Steven C. Almo ◽  
David G. Drubin
Keyword(s):  
Point Mutations ◽  
Fluid Phase ◽  
Intrinsic Rate ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
Cytoskeleton Organization

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin–cofilin generated during filament disassembly.

scholarly journals Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

1997 ◽  
Vol 136 (6) ◽  
pp. 1307-1322 ◽  
Author(s):  
Marie-France Carlier ◽  
Valérie Laurent ◽  
Jérôme Santolini ◽  
Ronald Melki ◽  
Dominique Didry ◽  
...  
Keyword(s):  
Actin Filament ◽  
Atp Hydrolysis ◽  
Fold Increase ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Filament Dynamics ◽  
Relevant Effect

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

scholarly journals Physical and functional interaction between the ID1 and p65 for activation of NF-κB

2012 ◽  
Vol 303 (3) ◽  
pp. C267-C277 ◽  
Author(s):  
Xiao Peng ◽  
Yuna Wang ◽  
Swapna Kolli ◽  
Junpeng Deng ◽  
Li Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Luciferase Assay ◽  
Physical Interaction ◽  
Site Mutation ◽  
Transcriptional Activation Domain ◽  
Apoptosis Protein

Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.

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