RESUMO
Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Forma Celular , Células Epiteliais/enzimologia , Guanilato Quinases/metabolismo , Junções Intercelulares/enzimologia , Proteína Quinase C/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Guanilato Quinases/genética , Células HEK293 , Homeostase , Humanos , Junções Intercelulares/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-2/genética , Proteína da Zônula de Oclusão-2/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: An immature intestine is a high-risk factor for necrotizing enterocolitis (NEC), which is a serious intestinal disease in newborns. The regulation of developmentally regulated GTP-binding protein 1 (DRG1) during organ development suggests a potential role of DRG1 in the maturation process of the intestine. AIM: To illustrate the function of DRG1 during the pathogenesis of NEC. METHODS: DRG1 expression in the intestine was measured using immunohistochemistry and q-PCR. Immunoprecipitation coupled with mass spectrometry was used to identify the interacting proteins of DRG1. The biological functions of the potential interactors were annotated with the Database for Annotation, Visualization and Integrated Discovery. Caco2 and FHs74Int cells with stable DRG1 silencing or overexpression were used to investigate the influence of DRG1 on cell junctions and intestinal barrier permeability and to elucidate the downstream mechanism. RESULTS: DRG1 was constitutively expressed during the intestinal maturation process but significantly decreased in the ileum in the context of NEC. Protein interaction analysis revealed that DRG1 was closely correlated with cell junctions. DRG1 deficiency destabilized the E-cadherin and occludin proteins near the cell membrane and increased the permeability of the epithelial cell monolayer, while DRG1 overexpression prevented lipopolysaccharide-induced disruption of E-cadherin and occludin expression and cell monolayer integrity. Further investigation suggested that DRG1 maintained cell junctions, especially adherens junctions, by regulating RAC1 activity, and RAC1 inhibition with NSC23766 attenuated intestinal injury and led to improved barrier integrity in experimental NEC. CONCLUSIONS: Our findings illustrate the mechanism underlying the effect of DRG1 deficiency on epithelial cell permeability regulation and provide evidence supporting the application of RAC1 inhibitors for protection against NEC.
Assuntos
Enterocolite Necrosante/enzimologia , Células Epiteliais/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Junções Intercelulares/enzimologia , Mucosa Intestinal/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Aminoquinolinas/farmacologia , Animais , Antígenos CD/metabolismo , Células CACO-2 , Caderinas/metabolismo , Impedância Elétrica , Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/genética , Junções Intercelulares/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Ocludina/metabolismo , Permeabilidade , Pirimidinas/farmacologia , Proteínas rac1 de Ligação ao GTP/análiseRESUMO
Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.
Assuntos
Adrenomedulina/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , Junções Intercelulares/efeitos dos fármacos , Linfangiogênese/efeitos dos fármacos , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Endotélio Linfático/enzimologia , Endotélio Linfático/patologia , Histamina/farmacologia , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/patologia , Camundongos , Camundongos Knockout , Permeabilidade , Transdução de Sinais , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Gut epithelium covers the inner layer of the gastrointestinal tract and provides a physical barrier to separate the host from its external environment, and its barrier function is critical for maintaining host health. AMP-activated protein kinase (AMPK) as a master regulator of energy metabolism plays a critical role in epithelial barrier function. AMPK activation promotes epithelial differentiation and facilitates cell polarity establishment, both of which strengthen epithelial barrier. In addition, AMPK promotes the assembly of tight junctions and adherens junctions by direct phosphorylation of proteins composing apical junctions, junctional anchors, and cytoskeletons. Pharmacological and nutraceutical compounds, as well as physiological states triggering AMPK activation strengthen epithelial barrier function. This review summarized recent progress in delineating the regulatory roles of AMPK in apical junction formation and barrier function of intestinal epithelium.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Junções Intercelulares/enzimologia , Mucosa Intestinal/enzimologia , Animais , HumanosRESUMO
Aims: With the maturation of placenta, ventricular chamber maturation enhances cardiac contractile performance to adapt to the metabolic demand of growing embryo. The organization of cardiomyocytes is required for the morphological remodelling in ventricular chamber maturation. However, the mechanism governing the establishment of cardiac cytoarchitecture during ventricular chamber maturation is still poorly studied. Methods and results: Here, we found that the expression of geranylgeranyl pyrophosphate synthase (Ggpps), which mediates protein geranylgeranylation, increased in the mouse heart after the onset of placental function. By using different Cre lines, we found that the cardiac inactivation of Ggpps by the Nkx2.5Cre/+ line disrupted protein geranylgeranylation as early as E9.5, which affected ventricular chamber maturation and resulted in mid-gestational embryonic lethality. In contrast, α-SMA-Cre line mediated the disruption of protein geranylgeranylation from E13.5 did not affect embryonic heart development. Further analysis of Nkx2.5Cre/+; Ggppsfl/fl mutants showed that the loss of Ggpps caused disorganized cardiac cytoarchitecture as early as E11.5 by disturbing cell-cell junctions. Ggpps inactivation decreased Rho GTPase geranylgeranylation and their activity, which might account for the disruption of cell-cell junctions. Moreover, elevating the protein geranylgeranylation by supplement of geranylgeranyl pyrophosphate (GGPP) could recover the Ggpps deficient induced defects of cytoarchitecture and cell-cell junctions in vitro and in vivo. Conclusion: Our present study demonstrates that GGPPS-mediated protein geranylgeranylation plays an indispensable role in the ventricular chamber maturation and acts as a stage-specific signal to regulate the establishment of cardiac cytoarchitecture during mid-gestation.
Assuntos
Farnesiltranstransferase/metabolismo , Coração Fetal/enzimologia , Complexos Multienzimáticos/metabolismo , Miócitos Cardíacos/enzimologia , Prenilação de Proteína , Animais , Farnesiltranstransferase/genética , Feminino , Coração Fetal/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Células HeLa , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Complexos Multienzimáticos/genética , Miócitos Cardíacos/ultraestrutura , Fenótipo , Gravidez , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression.
Assuntos
Caderinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Junções Intercelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Quinases Ativadas por p21/metabolismo , Antígenos CD , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/química , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Adesão Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/patologia , Gradação de Tumores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Bexiga Urinária/enzimologia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Urotélio/metabolismo , Urotélio/patologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/química , Quinases Ativadas por p21/genéticaRESUMO
AIMS: Loss-of-function mutations in the cytoskeletal protein ankyrin-B (AnkB) cause ventricular tachyarrhythmias in humans. Previously, we found that a larger fraction of the sarcoplasmic reticulum (SR) Ca(2+) leak occurs through Ca(2+) sparks in AnkB-deficient (AnkB(+/-)) mice, which may contribute to arrhythmogenicity via Ca(2+) waves. Here, we investigated the mechanisms responsible for increased Ca(2+) spark frequency in AnkB(+/-) hearts. METHODS AND RESULTS: Using immunoblots and phospho-specific antibodies, we found that phosphorylation of ryanodine receptors (RyRs) by CaMKII is enhanced in AnkB(+/-) hearts. In contrast, the PKA-mediated RyR phosphorylation was comparable in AnkB(+/-) and wild-type (WT) mice. CaMKII inhibition greatly reduced Ca(2+) spark frequency in myocytes from AnkB(+/-) mice but had little effect in the WT. Global activities of the major phosphatases PP1 and PP2A were similar in AnkB(+/-) and WT hearts, while CaMKII autophosphorylation, a marker of CaMKII activation, was increased in AnkB(+/-) hearts. Thus, CaMKII-dependent RyR hyperphosphorylation in AnkB(+/-) hearts is caused by augmented CaMKII activity. Intriguingly, CaMKII activation is limited to the sarcolemma-SR junctions since non-junctional CaMKII targets (phospholamban, HDAC4) are not hyperphosphorylated in AnkB(+/-) myocytes. This local CaMKII activation may be the consequence of elevated [Ca(2+)] in the junctional cleft caused by reduced Na(+)/Ca(2+) exchange activity. Indeed, using the RyR-targeted Ca(2+) sensor GCaMP2.2-FBKP12.6, we found that local junctional [Ca(2+)] is significantly elevated in AnkB(+/-) myocytes. CONCLUSIONS: The increased incidence of pro-arrhythmogenic Ca(2+) sparks and waves in AnkB(+/-) hearts is due to enhanced CaMKII-mediated RyR phosphorylation, which is caused by higher junctional [Ca(2+)] and consequent local CaMKII activation.
Assuntos
Anquirinas/deficiência , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/enzimologia , Animais , Anquirinas/genética , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Técnicas Biossensoriais , Ativação Enzimática , Genótipo , Junções Intercelulares/enzimologia , Potenciais da Membrana , Camundongos Knockout , Fenótipo , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimologia , Fatores de Tempo , Regulação para CimaRESUMO
Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury.
Assuntos
Inibidores de Calcineurina/farmacologia , Calcineurina/metabolismo , Junções Intercelulares/efeitos dos fármacos , Síndrome Nefrótica/tratamento farmacológico , Podócitos/efeitos dos fármacos , Proteinúria/tratamento farmacológico , Tacrolimo/farmacologia , Animais , Anticorpos Monoclonais , Linhagem Celular , Criança , Modelos Animais de Doenças , Feminino , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/patologia , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/congênito , Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/patologia , Podócitos/enzimologia , Podócitos/patologia , Transporte Proteico , Proteinúria/induzido quimicamente , Proteinúria/enzimologia , Proteinúria/patologia , Ratos Wistar , Fatores de TempoRESUMO
Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. The RhoA/Rho-kinase (ROCK) signaling pathway is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. Here we examined the potential role of this pathway in junctional zone (JZ) contraction in women with and without ADS. We demonstrated that in the normal JZ, RhoA and ROCK-I messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. Expression of RhoA and ROCK-I in the JZ from women with ADS was significantly higher than in the control women and showed no significant differences across the menstrual cycle. Treatment of JZ smooth muscle cells (JZSMCs) with estrogen at 0, 1, 10, or 100 nmol/L for 24 hours resulted in increased expression of RhoA, ROCK-I, and myosin light-chain (MLC) phosphorylation (p-MLC) in a dose-dependent manner. In parallel to its effects on p-MLC, estrogen-mediated, dose-dependent contraction responses in JZSMCs. Estrogen-mediated contraction in the ADS group was significantly higher than in the controls and also showed no significant differences across the menstrual cycle. These effects were suppressed in the presence of ICI 182780 or Y27632, supporting an estrogen receptor-dependent and RhoA activation-dependent mechanism. Our results indicate that the level of RhoA and ROCK-I increases in patients with ADS and the cyclic change is lost. Estrogen may affect uterine JZ contraction of ADS by enhancing RhoA/ ROCK-I signaling.
Assuntos
Adenomiose/enzimologia , Junções Intercelulares/enzimologia , Miócitos de Músculo Liso/enzimologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/biossíntese , Adenomiose/patologia , Adulto , Ativação Enzimática/fisiologia , Feminino , Humanos , Junções Intercelulares/patologia , Pessoa de Meia-Idade , Miócitos de Músculo Liso/patologiaRESUMO
The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.
Assuntos
Células Epiteliais/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Quinases Ativadas por p21/metabolismo , Sequência de Aminoácidos , Antígenos CD , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Junções Intercelulares/enzimologia , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response.
Assuntos
Permeabilidade Capilar , Células Endoteliais/enzimologia , Junções Intercelulares/enzimologia , Quinases Associadas a rho/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Junções Intercelulares/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais , Fibras de Estresse/enzimologia , Trombina/farmacologia , Fatores de Tempo , Transfecção , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
The bacterial pathogen Listeria monocytogenes uses actin-based motility to spread from infected human cells to surrounding healthy cells. Cell-cell spread involves the formation of thin extensions of the host plasma membrane ('protrusions') containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco-2 BBE1 induced a decrease in the level of Cdc42-GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen-induced downregulation of host Cdc42.
Assuntos
Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Células CACO-2 , Extensões da Superfície Celular/metabolismo , Regulação para Baixo , Repressão Enzimática , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/ultraestrutura , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/ultraestrutura , Listeriose/enzimologia , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity. METHODS AND RESULTS: Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity. CONCLUSIONS: In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.
Assuntos
Ácidos Heptanoicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Junções Intercelulares/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirróis/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Atorvastatina , Caderinas/genética , Caderinas/metabolismo , Guanosina Trifosfato/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Junções Intercelulares/enzimologia , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Lipopolissacarídeos/farmacologia , Ácido Mevalônico/farmacologia , Fosfatos de Poli-Isoprenil/farmacologia , Prenilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sesquiterpenos/farmacologia , Trombina/farmacologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
The ameloblast cell layer of the enamel organ is in contact with the forming enamel as it develops into the hardest substance in the body. Ameloblasts move in groups that slide by one another as the enamel layer thickens. Each ameloblast is responsible for the formation of one enamel rod, and the rods are the mineralized trail that moving ameloblasts leave behind. Matrix metalloproteinases (MMPs) facilitate cell movement in various tissues during development, and in this review we suggest that the tooth-specific MMP, enamelysin (MMP20), facilitates ameloblast movements during enamel development. Mmp20 null mice have thin brittle enamel with disrupted rod patterns that easily abrades from the underlying dentin. Strikingly, the Mmp20 null mouse enamel organ morphology is noticeably dysplastic during late-stage development, when MMP20 is no longer expressed. We suggest that in addition to its role of cleaving enamel matrix proteins, MMP20 also cleaves junctional complexes present on ameloblasts to foster the cell movement necessary for formation of the decussating enamel rod pattern. Therefore, inactivation of MMP20 would result in tight ameloblast cell-cell attachments that may cause maturation-stage enamel organ dysplasia. The tight ameloblast attachments would also preclude the ameloblast movement necessary to form decussating enamel rod patterns.
Assuntos
Ameloblastos/enzimologia , Junções Intercelulares/enzimologia , Metaloproteinase 20 da Matriz/fisiologia , Amelogênese/fisiologia , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Órgão do Esmalte/enzimologia , CamundongosRESUMO
The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K(d) = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr(1,380)-Phe(1,381)), thus defining a new mode of FERM domain-membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM-membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Junções Intercelulares/química , Junções Intercelulares/metabolismo , Animais , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/química , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células HEK293 , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/patologia , Proteína KRIT1 , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Glutathione transferase isozyme A4 (GSTA4) exhibits high catalytic efficiency to metabolize 4-hydroxynonenal (4-HNE), a highly reactive lipid peroxidation product that has been implicated in the pathogenesis of various chronic diseases. We investigated the role of 4-HNE in the mechanisms of unilateral ureteral obstruction (UUO)-induced fibrosis and its modulation by GSTA4-4 in a mouse model. Our data indicate that after UUO, accumulation of 4-HNE and its adducts were increased in renal tissues, with a concomitant decrease in the expression of GSTA4-4 in mice. As compared to wild-type (WT) mice, UUO caused an increased expression of fibroblast markers in the interstitium of GSTA4 KO mice. Additionally, increased autophagy and tubular cell damage were more severe in UUO-treated GSTA4 KO mice than in WT mice. Furthermore, GSK-3ß phosphorylation and expression of Snail, a regulator of E-cadherin and Occludin, was found to be significantly higher in UUO-inflicted GSTA4 KO mice. GSTA4 over-expression prevented 4-HNE-induced autophagy activation, tubular cell damage and Snail nuclear translocation in vitro. The effects of long-term expression of GSTA4 in restoration of UUO-induced damage in mice with the GSTA4 inducible transposon system indicated that release of obstruction after 3 days of UUO resulted in the attenuation of interstitial SMAα and collagen I expression. This transposon-delivered GSTA4 expression also suppressed UUO-induced loss of tubular cell junction markers and autophagy activation. Together, these results indicate that 4-HNE significantly contributes to the mechanisms of tubule injury and fibrosis and that these effects can be inhibited by the enhanced expression of GSTA4-4.
Assuntos
Glutationa Transferase/metabolismo , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Aldeídos/metabolismo , Animais , Autofagia/fisiologia , Células Cultivadas , Elementos de DNA Transponíveis , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose/enzimologia , Fibrose/patologia , Glutationa Transferase/genética , Junções Intercelulares/enzimologia , Junções Intercelulares/patologia , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos Knockout , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
The endothelium size selectively allows transport of fluids, ions, macromolecules, and leukocytes across the vessel wall paracellularly by dynamically opening intercellular junctions or transcellularly through caveolae. However, persistent opening of intercellular junctions leads to the formation of protein-rich edema in the interstitial tissue, a hallmark of tissue inflammation that, if left untreated, causes fatal diseases like acute respiratory distress syndrome (ARDS). The requirements for continuous transendothelial protein flux while limiting leukocyte flux into tissue imply that signaling processes exist in the endothelium that dynamically controls protein flux and leukocyte migration between the vascular and extravascular spaces. In this chapter, we discuss the signaling mechanisms elicited by several well-known inflammatory mediators that increase endothelial permeability. Specifically, we have concentrated in reviewing the new concepts dealing with the restoration of normal endothelial permeability by sphingosine kinase 1 following inflammatory stimulus.
Assuntos
Permeabilidade Capilar/fisiologia , Animais , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Humanos , Junções Intercelulares/enzimologia , Junções Intercelulares/metabolismo , Liases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Transdução de SinaisRESUMO
Apical-domain constriction is important for regulating epithelial morphogenesis. Epithelial cells are connected by apical junctional complexes (AJCs) that are lined with circumferential actomyosin cables. The contractility of these cables is regulated by Rho-associated kinases (ROCKs). Here, we report that Willin (a FERM-domain protein) and Par3 (a polarity-regulating protein) cooperatively regulate ROCK-dependent apical constriction. We found that Willin recruits aPKC and Par6 to the AJCs, independently of Par3. Simultaneous depletion of Willin and Par3 completely removed aPKC and Par6 from the AJCs and induced apical constriction. Induced constriction was through upregulation of the level of AJC-associated ROCKs, which was due to loss of aPKC. Our results indicate that aPKC phosphorylates ROCK and suppresses its junctional localization, thereby allowing cells to retain normally shaped apical domains. Thus, we have uncovered a Willin/Par3-aPKC-ROCK pathway that controls epithelial apical morphology.
Assuntos
Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Forma Celular , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/enzimologia , Junções Intercelulares/enzimologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Quinases Associadas a rho/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto/genética , Cães , Células HEK293 , Humanos , Isoenzimas/genética , Camundongos , Mutação , Proteínas do Tecido Nervoso , Fosforilação , Proteína Quinase C/genética , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Quinases Associadas a rho/genéticaRESUMO
The regulation of adherens junctions (AJs) is critical for multiple events during CNS development, including the formation and maintenance of the neuroepithelium. We have addressed the role of the small GTPase RhoA in the developing mouse nervous system using tissue-specific conditional gene ablation. We show that, in the spinal cord neuroepithelium, RhoA is essential to localize N-cadherin and ß-catenin to AJs and maintain apical-basal polarity of neural progenitor cells. Ablation of RhoA caused the loss of AJs and severe abnormalities in the organization of cells within the neuroepithelium, including decreased neuroepithelial cell proliferation and premature cell-cycle exit, reduction of the neural stem cell pool size, and the infiltration of neuroepithelial cells into the lumen of the ventricle. We also show that, in the absence of RhoA, its effector, mammalian diaphanous-related formin1 (mDia1), does not localize to apical AJs in which it likely stabilizes intracellular adhesion by promoting local actin polymerization and microtubule organization. Furthermore, expressing a dominant-negative form of mDia1 in neural stem/progenitor cells results in a similar phenotype compared with that of the RhoA conditional knock-out, namely the loss of AJs and apical polarity. Together, our data show that RhoA signaling is necessary for AJ regulation and for the maintenance of mammalian neuroepithelium organization preventing precocious cell-cycle exit and differentiation.
Assuntos
Células-Tronco Neurais/citologia , Células-Tronco Neurais/enzimologia , Células Neuroepiteliais/enzimologia , Medula Espinal/enzimologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Feminino , Junções Intercelulares/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Células Neuroepiteliais/citologia , Neurogênese/fisiologia , Gravidez , Transdução de Sinais/fisiologia , Medula Espinal/citologiaRESUMO
A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.