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1.
J Clin Invest ; 134(13)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38768074

RESUMO

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, DSP-/- EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-κB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.


Assuntos
Imunidade Inata , Células-Tronco Pluripotentes Induzidas , Miocardite , Miócitos Cardíacos , Humanos , Miocardite/genética , Miocardite/imunologia , Miocardite/patologia , Imunidade Inata/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Masculino , Predisposição Genética para Doença , Feminino
2.
Sci Rep ; 11(1): 13295, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168237

RESUMO

Desmosomes have a central role in mediating extracellular adhesion between cells, but they also coordinate other biological processes such as proliferation, differentiation, apoptosis and migration. In particular, several lines of evidence have implicated desmosomal proteins in regulating the actin cytoskeleton and attachment to the extracellular matrix, indicating signaling crosstalk between cell-cell junctions and cell-matrix adhesions. In our study, we found that cells lacking the desmosomal cadherin Desmoglein-2 (Dsg2) displayed a significant increase in spreading area on both fibronectin and collagen, compared to control A431 cells. Intriguingly, this effect was observed in single spreading cells, indicating that Dsg2 can exert its effects on cell spreading independent of cell-cell adhesion. We hypothesized that Dsg2 may mediate cell-matrix adhesion via control of Rap1 GTPase, which is well known as a central regulator of cell spreading dynamics. We show that Rap1 activity is elevated in Dsg2 knockout cells, and that Dsg2 harnesses Rap1 and downstream TGFß signaling to influence both cell spreading and focal adhesion protein phosphorylation. Further analysis implicated the Rap GEF PDZ-GEF2 in mediating Dsg2-dependent cell spreading. These data have identified a novel role for Dsg2 in controlling cell spreading, providing insight into the mechanisms via which cadherins exert non-canonical junction-independent effects.


Assuntos
Adesão Celular , Desmogleína 2/metabolismo , Adesões Focais/metabolismo , Western Blotting , Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
3.
J Invest Dermatol ; 139(6): 1227-1236, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30579854

RESUMO

Desmoplakin (DP) is an obligate component of desmosomal cell-cell junctions that links the adhesion plaque to the cytoskeletal intermediate filament network. While a central role for DP in maintaining the structure and stability of the desmosome is well established, recent work has indicated that DP's functions may extend beyond cell-cell adhesion. In our study, we show that loss of DP results in a significant increase in cellular migration, as measured by scratch wound assays, Transwell migration assays, and invasion assays. Loss of DP causes dramatic changes in actin cytoskeleton morphology, including enhanced protrusiveness, and an increase in filopodia length and number. Interestingly, these changes are also observed in single cells, indicating that control of actin morphology is a cell-cell adhesion-independent function of DP. An investigation of cellular signaling pathways uncovered aberrant Rac and p38 mitogen-activated protein kinase (MAPK) activity in DP knockdown cells, restoration of which is sufficient to rescue DP-dependent changes in both cell migration and actin cytoskeleton morphology. Taken together, these data highlight a previously uncharacterized role for the desmosomal cytolinker DP in coordinating cellular migration via p38 MAPK and Rac signaling.


Assuntos
Movimento Celular/fisiologia , Desmoplaquinas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Amidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desmoplaquinas/genética , Técnicas de Silenciamento de Genes , Humanos , Filamentos Intermediários/metabolismo , Piridinas/farmacologia , Pironas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Zearalenona/análogos & derivados , Zearalenona/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
4.
J Cell Biol ; 217(9): 3219-3235, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29959233

RESUMO

Desmoplakin (DP) is an obligate component of desmosomes, intercellular adhesive junctions that maintain the integrity of the epidermis and myocardium. Mutations in DP can cause cardiac and cutaneous disease, including arrhythmogenic cardiomyopathy (ACM), an inherited disorder that frequently results in deadly arrhythmias. Conduction defects in ACM are linked to the remodeling and functional interference with Cx43-based gap junctions that electrically and chemically couple cells. How DP loss impairs gap junctions is poorly understood. We show that DP prevents lysosomal-mediated degradation of Cx43. DP loss triggered robust activation of ERK1/2-MAPK and increased phosphorylation of S279/282 of Cx43, which signals clathrin-mediated internalization and subsequent lysosomal degradation of Cx43. RNA sequencing revealed Ras-GTPases as candidates for the aberrant activation of ERK1/2 upon loss of DP. Using a novel Ras inhibitor, Ras/Rap1-specific peptidase (RRSP), or K-Ras knockdown, we demonstrate restoration of Cx43 in DP-deficient cardiomyocytes. Collectively, our results reveal a novel mechanism for the regulation of the Cx43 life cycle by DP in cardiocutaneous models.


Assuntos
Conexina 43/metabolismo , Desmoplaquinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Junções Comunicantes/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Cardiomiopatias/patologia , Comunicação Celular/fisiologia , Células Cultivadas , Clatrina/metabolismo , Desmoplaquinas/genética , Desmossomos/fisiologia , Ativação Enzimática/genética , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Ratos Sprague-Dawley
5.
J Cell Biol ; 212(4): 425-38, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26858265

RESUMO

Members of the desmosome protein family are integral components of the cardiac area composita, a mixed junctional complex responsible for electromechanical coupling between cardiomyocytes. In this study, we provide evidence that loss of the desmosomal armadillo protein Plakophilin-2 (PKP2) in cardiomyocytes elevates transforming growth factor ß1 (TGF-ß1) and p38 mitogen-activated protein kinase (MAPK) signaling, which together coordinate a transcriptional program that results in increased expression of profibrotic genes. Importantly, we demonstrate that expression of Desmoplakin (DP) is lost upon PKP2 knockdown and that restoration of DP expression rescues the activation of this TGF-ß1/p38 MAPK transcriptional cascade. Tissues from PKP2 heterozygous and DP conditional knockout mouse models also exhibit elevated TGF-ß1/p38 MAPK signaling and induction of fibrotic gene expression in vivo. These data therefore identify PKP2 and DP as central players in coordination of desmosome-dependent TGF-ß1/p38 MAPK signaling in cardiomyocytes, pathways known to play a role in different types of cardiac disease, such as arrhythmogenic or hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatias/enzimologia , Miócitos Cardíacos/enzimologia , Placofilinas/deficiência , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Cardiomiopatias/genética , Cardiomiopatias/patologia , Linhagem Celular , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Ativação Enzimática , Fibrose , Regulação da Expressão Gênica , Genótipo , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Fenótipo , Fosforilação , Placofilinas/genética , Estabilidade Proteica , Interferência de RNA , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção
6.
J Cell Biol ; 206(6): 779-97, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25225338

RESUMO

Mechanisms by which microtubule plus ends interact with regions of cell-cell contact during tissue development and morphogenesis are not fully understood. We characterize a previously unreported interaction between the microtubule binding protein end-binding 1 (EB1) and the desmosomal protein desmoplakin (DP), and demonstrate that DP-EB1 interactions enable DP to modify microtubule organization and dynamics near sites of cell-cell contact. EB1 interacts with a region of the DP N terminus containing a hotspot for pathogenic mutations associated with arrhythmogenic cardiomyopathy (AC). We show that a subset of AC mutations, in addition to a mutation associated with skin fragility/woolly hair syndrome, impair gap junction localization and function by misregulating DP-EB1 interactions and altering microtubule dynamics. This work identifies a novel function for a desmosomal protein in regulating microtubules that affect membrane targeting of gap junction components, and elucidates a mechanism by which DP mutations may contribute to the development of cardiac and cutaneous diseases.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Conexina 43/metabolismo , Desmoplaquinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Células COS , Comunicação Celular , Linhagem Celular , Chlorocebus aethiops , Demecolcina/farmacologia , Desmogleína 2/genética , Desmoplaquinas/genética , Desmossomos/fisiologia , Junções Comunicantes/genética , Junções Comunicantes/patologia , Células HEK293 , Humanos , Morfogênese , Mutação , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Moduladores de Tubulina/farmacologia
7.
J Cell Biol ; 202(4): 653-66, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23940119

RESUMO

Although much is known about signaling factors downstream of Rho GTPases that contribute to epidermal differentiation, little is known about which upstream regulatory proteins (guanine nucleotide exchange factors [GEFs] or GTPase-activating proteins [GAPs]) are involved in coordinating Rho signaling in keratinocytes. Here we identify the GEF breakpoint cluster region (Bcr) as a major upstream regulator of RhoA activity, stress fibers, and focal adhesion formation in keratinocytes. Loss of Bcr reduced expression of multiple markers of differentiation (such as desmoglein-1 [Dsg1], keratin-1, and loricrin) and abrogated MAL/SRF signaling in differentiating keratinocytes. We further demonstrated that loss of Bcr or MAL reduced levels of Dsg1 mRNA in keratinocytes, and ectopic expression of Dsg1 rescued defects in differentiation seen upon loss of Bcr or MAL signaling. Taken together, these data identify the GEF Bcr as a regulator of RhoA/MAL signaling in keratinocytes, which in turn promotes differentiation through the desmosomal cadherin Dsg1.


Assuntos
Diferenciação Celular , Desmogleína 1/metabolismo , Queratinócitos/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Humanos , Queratinócitos/citologia , RNA Mensageiro/metabolismo
8.
J Clin Invest ; 123(4): 1556-70, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23524970

RESUMO

Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Desmogleína 1/metabolismo , Epiderme/patologia , Queratinócitos/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Células Cultivadas , Desmocolinas/metabolismo , Desmogleína 1/genética , Desmogleína 1/fisiologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Ceratodermia Palmar e Plantar/metabolismo , Ceratodermia Palmar e Plantar/patologia , Laminas/genética , Laminas/metabolismo , Masculino , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , RNA Interferente Pequeno/genética , Adulto Jovem , Proteínas ras/metabolismo
9.
Nat Protoc ; 6(12): 2050-60, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22134128

RESUMO

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.


Assuntos
Núcleo Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/análise , Proteína rhoA de Ligação ao GTP/análise , Fracionamento Celular/métodos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho , Frações Subcelulares/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
11.
PLoS One ; 6(2): e17380, 2011 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-21390328

RESUMO

BACKGROUND: Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells. PRINCIPAL FINDINGS: Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA/fisiologia , Proteínas Oncogênicas/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Distribuição Tecidual/efeitos dos fármacos
12.
J Cell Sci ; 123(Pt 20): 3576-86, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20876660

RESUMO

We previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG⁻/⁻ cells onto ECM deposited by PG+/⁻ cells partially restored normal cell morphology and inhibited PG⁻/⁻ cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PG⁻/⁻ cells, a substantial decrease in fibronectin (FN) in PG⁻/⁻ cells stood out. Re-introduction of PG into PG⁻/⁻ cells restored FN expression, and keratinocyte motility was reversed by plating PG⁻/⁻ cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG⁻/⁻ cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG⁻/⁻ cells on ECM deposited by PG+/⁻ keratinocytes. PG⁻/⁻ cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG⁻/⁻ keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.


Assuntos
Movimento Celular/fisiologia , Fibronectinas/metabolismo , Transdução de Sinais/fisiologia , gama Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Movimento Celular/genética , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Técnica Indireta de Fluorescência para Anticorpo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , gama Catenina/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética
13.
Arterioscler Thromb Vasc Biol ; 30(9): 1779-86, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20702813

RESUMO

OBJECTIVE: The goals of this study were to identify the signaling pathway by which sphingosine 1-phosphate (S1P) activates RhoA in smooth muscle cells (SMC) and to evaluate the contribution of this pathway to the regulation of SMC phenotype. METHODS AND RESULTS: Using a combination of receptor-specific agonists and antagonists we identified S1P receptor 2 (S1PR2) as the major S1P receptor subtype that regulates SMC differentiation marker gene expression. Based on the known coupling properties of S1PR2 and our demonstration that overexpression of Galpha(12) or Galpha(13) increased SMC-specific promoter activity, we next tested whether the effects of S1P in SMC were mediated by the regulator of G protein-signaling-Rho guanine exchange factors (RGS-RhoGEFs) (leukemia-associated RhoGEF [LARG], PDZ-RhoGEF [PRG], RhoGEF [p115]). Although each of the RGS-RhoGEFs enhanced actin polymerization, myocardin-related transcription factor-A nuclear localization, and SMC-specific promoter activity when overexpressed in 10T1/2 cells, LARG exhibited the most robust effect and was the only RGS-RhoGEF activated by S1P in SMC. Importantly, siRNA-mediated depletion of LARG significantly inhibited the activation of RhoA and SMC differentiation marker gene expression by S1P. Knockdown of LARG had no effect on SMC proliferation but promoted SMC migration as measured by scratch wound and transwell assays. CONCLUSIONS: These data indicate that S1PR2-dependent activation of RhoA in SMC is mediated by LARG and that this signaling mechanism promotes the differentiated SMC phenotype.


Assuntos
Diferenciação Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lisofosfolipídeos/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular , Ativação Enzimática , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Fenótipo , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína rhoA de Ligação ao GTP
14.
Mol Biol Cell ; 21(16): 2844-59, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20554761

RESUMO

Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.


Assuntos
Actomiosina/metabolismo , Desmossomos/metabolismo , Placofilinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Caderinas/metabolismo , Cateninas/metabolismo , Comunicação Celular , Linhagem Celular , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/metabolismo , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Placofilinas/genética , Ligação Proteica , Proteína Quinase C/metabolismo , Interferência de RNA , Transdução de Sinais , delta Catenina
15.
Int Rev Cell Mol Biol ; 277: 1-65, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19766966

RESUMO

Focal adhesions have been intensely studied ever since their discovery in 1971. The last three decades have seen major advances in understanding the structure of focal adhesions and the functions they serve in cellular adhesion, migration, and other biological processes. In this chapter, we begin with a historical perspective of focal adhesions, provide an overview of focal adhesion biology, and highlight recent major advances in the field. Specifically, we review the different types of matrix adhesions and the role different Rho GTPases play in their formation. We discuss the relative contributions of integrin and syndecan adhesion receptors to the formation of focal adhesions. We also focus on new insights gained from studying focal adhesions on biomaterial surfaces and from the growing field of mechanotransduction. Throughout this chapter, we have highlighted areas of focal adhesion biology where major questions still remain to be answered.


Assuntos
Adesões Focais/metabolismo , Animais , Humanos , Receptores de Superfície Celular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
16.
J Cell Sci ; 120(Pt 22): 3989-98, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17971419

RESUMO

Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.


Assuntos
Fibroblastos/citologia , Fibroblastos/enzimologia , Fibronectinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Transporte Proteico , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Fibras de Estresse/metabolismo
17.
Mol Cell Biol ; 27(24): 8683-97, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17938206

RESUMO

Net1 is a RhoA-specific guanine nucleotide exchange factor which localizes to the nucleus at steady state. A deletion in its N terminus redistributes the protein to the cytosol, where it activates RhoA and can promote transformation. Net1 contains a PDZ-binding motif at the C terminus which is essential for its transformation properties. Here, we found that Net1 interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP102, and PSD95. The interaction between Net1 and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. Interestingly, the oncogenic mutant of Net1 is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. Our results suggest that the ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. In agreement with this, the transformation potential of oncogenic Net1 is reduced when it is coexpressed with Dlg1 or SAP102. Together, our results suggest that the interaction between Net1 and Dlg1 may contribute to the mechanism of Net1-mediated transformation.


Assuntos
Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Transformação Celular Neoplásica , Citosol/metabolismo , Proteína 1 Homóloga a Discs-Large , Proteína 4 Homóloga a Disks-Large , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Sinais de Localização Nuclear , Proteínas Oncogênicas/química , Domínios PDZ , Ligação Proteica , Transporte Proteico , Ratos , Proteínas Associadas SAP90-PSD95 , Deleção de Sequência
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