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Cell adhesion and migration depend on the assembly and disassembly of adhesive structures known as focal adhesions. Cells adhere to the extracellular matrix (ECM) and form these structures via receptors, such as integrins and syndecans, which initiate signal transduction pathways that bridge the ECM to the cytoskeleton, thus governing adhesion and migration processes. Integrins bind to the ECM and soluble or cell surface ligands to form integrin adhesion complexes (IAC), whose composition depends on the cellular context and cell type. Proteomic analyses of these IACs led to the curation of the term adhesome, which is a complex molecular network containing hundreds of proteins involved in signaling, adhesion, and cell movement. One of the hallmarks of these IACs is to sense mechanical cues that arise due to ECM rigidity, as well as the tension exerted by cell-cell interactions, and transduce this force by modifying the actin cytoskeleton to regulate cell migration. Among the integrin/syndecan cell surface ligands, we have described Thy-1 (CD90), a GPI-anchored protein that possesses binding domains for each of these receptors and, upon engaging them, stimulates cell adhesion and migration. In this review, we examine what is currently known about adhesomes, revise how mechanical forces have changed our view on the regulation of cell migration, and, in this context, discuss how we have contributed to the understanding of signaling mechanisms that control cell adhesion and migration.
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Cell migration is essential for many biological and pathological processes. Establishing cell polarity with a trailing edge and forming a single lamellipodium at the leading edge of the cell is crucial for efficient directional cell migration and is a hallmark of mesenchymal cell motility. Lamellipodia formation is regulated by spatial-temporal activation of the small GTPases Rac and Cdc42 at the front edge, and RhoA at the rear end. At a molecular level, partitioning-defective (Par) protein complex comprising Par3, Par6, and atypical Protein Kinase (aPKC isoforms ζ and λ/ι) regulates front-rear axis polarization. At the front edge, integrin clustering activates Cdc42, prompting the formation of Par3/Par6/aPKC complexes to modulate MTOC positioning and microtubule stabilization. Consequently, the Par3/Par6/aPKC complex recruits Rac1-GEF Tiam to activate Rac1, leading to lamellipodium formation. At the rear end, RhoA-ROCK phosphorylates Par3 disrupting its interaction with Tiam and inactivating Rac1. RhoA activity at the rear end allows the formation of focal adhesions and stress fibers necessary to generate the traction forces that allow cell movement. Nox1-based NADPH oxidase is necessary for PDGF-induced migration in vitro and in vivo for many cell types, including fibroblasts and smooth muscle cells. Here, we report that Nox1-deficient cells failed to acquire a normal front-to-rear polarity, polarize MTOC, and form a single lamellipodium. Instead, these cells form multiple protrusions that accumulate Par3 and active Tiam. The exogenous addition of H2O2 rescues this phenotype and is associated with the hyperactivation of Par3, Tiam, and Rac1. Mechanistically, Nox1 deficiency induces the inactivation of PP2A phosphatase, leading to increased activation of aPKC. These results were validated in Nox1y/- primary mouse aortic smooth muscle cells (MASMCs), which also showed PP2A inactivation after PDGF-BB stimulation consistent with exacerbated activation of aPKC. Moreover, we evaluated the physiological relevance of this signaling pathway using a femoral artery wire injury model to generate neointimal hyperplasia. Nox1y/- mice showed increased staining for the inactive form of PP2A and increased signal for active aPKC, suggesting that PP2A and aPKC activities might contribute to reducing neointima formation observed in the arteries of Nox1y/- mice.
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Background Lung injury, a severe adverse outcome of lipopolysaccharide-induced acute respiratory distress syndrome, is attributed to excessive neutrophil recruitment and effector response. Poldip2 (polymerase δ-interacting protein 2) plays a critical role in regulating endothelial permeability and leukocyte recruitment in acute inflammation. Thus, we hypothesized that myeloid Poldip2 is involved in neutrophil recruitment to inflamed lungs. Methods and Results After characterizing myeloid-specific Poldip2 knockout mice, we showed that at 18 hours post-lipopolysaccharide injection, bronchoalveolar lavage from myeloid Poldip2-deficient mice contained fewer inflammatory cells (8 [4-16] versus 29 [12-57]×104/mL in wild-type mice) and a smaller percentage of neutrophils (30% [28%-34%] versus 38% [33%-41%] in wild-type mice), while the main chemoattractants for neutrophils remained unaffected. In vitro, Poldip2-deficient neutrophils responded as well as wild-type neutrophils to inflammatory stimuli with respect to neutrophil extracellular trap formation, reactive oxygen species production, and induction of cytokines. However, neutrophil adherence to a tumor necrosis factor-α stimulated endothelial monolayer was inhibited by Poldip2 depletion (225 [115-272] wild-type [myePoldip2+/+] versus 133 [62-178] myeloid-specific Poldip2 knockout [myePoldip2-/-] neutrophils) as was transmigration (1.7 [1.3-2.1] versus 1.1 [1.0-1.4] relative to baseline transmigration). To determine the underlying mechanism, we examined the surface expression of ß2-integrin, its binding to soluble intercellular adhesion molecule 1, and Pyk2 phosphorylation. Surface expression of ß2-integrins was not affected by Poldip2 deletion, whereas ß2-integrins and Pyk2 were less activated in Poldip2-deficient neutrophils. Conclusions These results suggest that myeloid Poldip2 is involved in ß2-integrin activation during the inflammatory response, which in turn mediates neutrophil-to-endothelium adhesion in lipopolysaccharide-induced acute respiratory distress syndrome.
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Proteínas Mitocondriales , Neutrófilos , Proteínas Nucleares , Neumonía , Síndrome de Dificultad Respiratoria , Animales , Adhesión Celular , Modelos Animales de Enfermedad , Quinasa 2 de Adhesión Focal/metabolismo , Integrinas/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neumonía/genética , Neumonía/metabolismo , Neumonía/patología , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
Acute skin wound healing is a multistage process consisting of a plethora of tightly regulated signaling events in specialized cells. The Thy-1 (CD90) glycoprotein interacts with integrins and the heparan sulfate proteoglycan syndecan 4, generating a trimolecular complex that triggers bi-directional signaling to regulate diverse aspects of the wound healing process. These proteins can act either as ligands or receptors, and they are critical for the successful progression of wound healing. The expression of Thy-1, integrins, and syndecan 4 is controlled during the healing process, and the lack of expression of any of these proteins results in delayed wound healing. Here, we review and discuss the roles and regulatory events along the stages of wound healing that support the relevance of Thy-1, integrins, and syndecan 4 as crucial regulators of skin wound healing.
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AIMS: Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2. METHODS AND RESULTS: Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC-/- mice showed reduced lung leucocyte infiltration in BAL (0.21 ± 0.9×106 vs. 1.29 ± 1.8×106 cells/mL) and lung tissue (12.7 ± 1.8 vs. 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs. 4.15 ± 0.5-fold, IκBα 4.32 ± 1.53 vs. 8.97 ± 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs. 147 ± 25.7-fold, CXCL2 65 ± 25.6 vs. 215 ± 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs. 3.8 ± 0.38-fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 ± 0.5 vs. 17.5 ± 3-fold for permeability, 1.5 ± 0.4 vs. 10.9 ± 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs. 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell. CONCLUSION: Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.
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Lesión Pulmonar , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/metabolismo , Sepsis , Animales , Endotelio/metabolismo , Humanos , Pulmón/metabolismo , Lesión Pulmonar/genética , Ratones , Proteínas Mitocondriales/genética , Proteínas Nucleares/genética , Sepsis/complicaciones , Sepsis/genética , Sepsis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Background The growth and remodeling of vascular networks is an important component of the prognosis for patients with peripheral artery disease. One protein that has been previously implicated to play a role in this process is RAGE (receptor for advanced glycation end products). This study sought to determine the cellular source of RAGE in the ischemic hind limb and the role of RAGE signaling in this cell type. Methods and Results Using a hind limb ischemia model of vascular growth, this study found skeletal muscle satellite cells to be a novel major cellular source of RAGE in ischemic tissue by both staining and cellular sorting. Although wild-type satellite cells increased tumor necrosis factor-α and monocyte chemoattractant protein-1 production in response to ischemia in vivo and a RAGE ligand in vitro, satellite cells from RAGE knockout mice lacked the increase in cytokine production both in vivo in response to ischemia and in vitro after stimuli with the RAGE ligand high-mobility group box 1. Furthermore, encapsulated wild-type satellite cells improved perfusion after hind limb ischemia surgery by both perfusion staining and vessel quantification, but RAGE knockout satellite cells provided no improvement over empty capsules. Conclusions Thus, RAGE expression and signaling in satellite cells is crucial for their response to stimuli and angiogenic and arteriogenic functions.
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Productos Finales de Glicación Avanzada , Isquemia , Animales , Miembro Posterior , Humanos , Isquemia/genética , Ligandos , Ratones , Receptor para Productos Finales de Glicación Avanzada/genéticaRESUMEN
This study consisted of developing a dressing loaded with silver (Ag) and ibuprofen (IBU) that provides a dual therapy, antibacterial and antalgic, intended for infected painful wounds. Therefore, non-woven polyethyleneterephtalate (PET) textiles nonwovens were pre-treated by cyclodextrin crosslinked with citric acid by a pad/dry/cure process. Then, textiles were impregnated in silver solution followed by a thermal treatment and were then coated by Layer-by-Layer (L-b-L) deposition of a polyelectrolyte multilayer (PEM) system consisting of anionic water-soluble poly(betacyclodextrin citrate) (PCD) and cationic chitosan. Finally, ibuprofen lysinate (IBU-L) was loaded on the PEM coating. We demonstrated the complexation of IBU with native ßCD and PCD by phase solubility diagram and 1H NMR. PEM system allowed complete IBU-L release in 6 h in PBS pH 7.4 batch (USP IV). On the other hand, microbiological tests demonstrated that loaded silver induced bacterial reduction of 4 Log10 against S. aureus and E. coli and tests revealed that ibuprofen lysinate loading did not interfere with the antibacterial properties of the dressing.
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The current pandemic caused by SARS-CoV-2 has put public health at risk, being wastewater-based epidemiology (WBE) a potential tool in the detection, prevention, and treatment of present and possible future outbreaks, since this virus enters wastewater through various sources such as feces, vomit, and sputum. Thus, advanced technologies such as advanced oxidation processes (AOP), membrane technology (MT) are identified through a systematic literature review as an alternative option for the destruction and removal of emerging contaminants (drugs and personal care products) released mainly by infected patients. The objectives of this review are to know the implications that the new COVID-19 outbreak is generating and will generate in water compartments, as well as the new challenges faced by wastewater treatment plants due to the change in a load of contaminants and the solutions proposed based on the aforementioned technologies to be applied to preserve public health and the environment.
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BACKGROUND: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvß3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adhesiones Focales/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Sindecano-4/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Adhesión Celular , Línea Celular , Movimiento Celular , Polaridad Celular , Fibroblastos/metabolismo , Silenciador del Gen , Ratones , Microtúbulos/metabolismo , Unión Proteica , Ratas , Antígenos Thy-1/metabolismoRESUMEN
AIMS: Gynaecological malignancies such as breast, ovarian and cervical cancers have become an important public health problem. Detection of molecular alterations in cancer research is fundamental since it can reveal specific pathogenic patterns and genes that could serve as markers. Our aim was to characterize common genomic and transcriptomic signatures for the three gynaecologic malignancies with the highest incidence and mortality to try to identify new molecular markers, therapeutic targets and molecular signatures. METHODS: Here we analysed a total of 723 microarray libraries corresponding to equal number of breast, ovary and cervical cancer and non-cancer patient samples. Copy number variation (CNV) was carried out using 428 libraries and transcriptomic analysis using the 295 remaining samples. RESULTS: Our results showed that breast, ovary and cervical malignancies are characterized by gain of 1q chromosome. At transcriptomic level, they share 351 coding and non-coding genes, which could represent core transcriptome of gynaecological malignancies. Pathway analysis from the resulting gene lists from CNV and expression showed participation in cell cycle, metabolism, and cell adhesion molecules among others. CONCLUSIONS: Chromosome 1q characterize the gynaecological malignancies, which could harbour a richness of genetic repertoire to mine for molecular markers and targets, particular gynaecologic expression profile, containing FANCI, FH and MIR155HG among others, could represent part of the transcriptomic core for diagnostic test and attractive therapeutic targets. It may not be long before every human cancer sample is profiled for a detections test to ascertain a molecular diagnosis and prognosis and to define an optimal and precise treatment strategy.
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Biomarcadores/metabolismo , Perfilación de la Expresión Génica/métodos , Neoplasias de los Genitales Femeninos/economía , Neoplasias de los Genitales Femeninos/genética , Genómica/métodos , Medicina de Precisión/métodos , Transcriptoma/genética , Femenino , Humanos , PronósticoRESUMEN
The aim of this work is to design a wound dressing able to release chlorhexidine (CHX) as antiseptic agent, ensuring long-lasting antibacterial efficacy during the healing. The textile nonwoven (polyethylene terephthalate) (PET) of the dressing was first modified by chitosan (CHT) crosslinked with genipin (Gpn). Parameters such as the concentration of reagents (Gpn and CHT) but also the crosslinking time and the working temperature were optimized to reach the maximal positive charges surface density. This support was then treated by the layer-by-layer (LbL) deposition of a multilayer system composed of methyl-beta-cyclodextrin polymer (PCD) (anionic) and CHT (cationic). After a thermal treatment to stabilize the LbL film, the textiles were loaded with CHX as antiseptic agent. The influence of the thermal treatment i) on the cytocompatibility, ii) on the degradation of the multilayer system, iii) on CHX sorption and release profiles and iv) on the antibacterial activity of the loaded textiles was studied.
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Antibacterianos/farmacología , Vendajes , Clorhexidina/farmacología , Materiales Biocompatibles Revestidos/farmacología , Textiles , Cicatrización de Heridas/efectos de los fármacos , Adsorción , Línea Celular , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/química , Liberación de Fármacos , Calor , Humanos , Iridoides/farmacología , Pruebas de Sensibilidad Microbiana , Polielectrolitos/química , Tereftalatos Polietilenos/química , Solubilidad , Staphylococcus aureus/efectos de los fármacos , Factores de TiempoRESUMEN
The goal of the study was to elaborate an antibacterial silver wound dressing covered by a protective coating that would prevent silver diffusion toward skin without losing its biocide properties. Therefore, non woven polyethyleneterephtalate (PET) textiles were pre-treated by two types of polysaccharides - chitosan and cyclodextrin - both crosslinked with citric acid by a pad/dry/cure process. Both types of resulting thermofixed textiles carrying the citrate crosslinks were then impregnated in silver solution followed by a thermal treatment and were finally coated by Layer-by-Layer (L-b-L) deposition of a polyelectrolyte multilayer (PEM) film consisting of anionic water-soluble poly-cyclodextrin and cationic chitosan. The influence of the process parameters was investigated in terms of silver adsorption capacity, PEM system build-up, silver kinetics of release and antibacterial activity. We demonstrate i) the utility of the intermediate thermal treatment step in the reduction of silver leakage in the polyelectrolyte solutions used in the L-b-L process, ii) that silver adsorption on the preliminary thermofixed layers did not affect the PEM system build-up, iii) the slowing down of silver release kinetic thanks to the PEM coating, iv) the preservation of the antibacterial activity despite the PEM coating.
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Antibacterianos/administración & dosificación , Química Farmacéutica/métodos , Compuestos de Plata/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Antibacterianos/farmacología , Quitosano/química , Ácido Cítrico/química , Reactivos de Enlaces Cruzados/química , Ciclodextrinas/química , Liberación de Fármacos , Polielectrolitos/química , Compuestos de Plata/farmacologíaRESUMEN
Introduction: In Head and Neck (HN) cancer, the High-Risk Human Papillomavirus (hr HPV) infection has been associated in about 40% of these tumors. The hr HPV infection is one of the etiological factors of several epithelial tumors; however, its association with the prognosis has not yet been established for patients with Laryngeal Squamous Cell Carcinoma (LSCC). On the other hand, Epidermal Growth Factor Receptor (EGFR) is a molecular marker widely studied in cancer and its overexpression has been associated with poor prognosis in some types of cancer, including the HN cancer. In the present study, we analyzed EGFR expression and HPV detection in a cohort of Mexican patients with LSCC and define their association with clinical-pathological and survival parameters. Methods: EGFR expression analysis was performed by immunohistochemistry assay. A tissue array was constructed based on 30 paraffin-embedded tissue samples. HPV detection was performed by PCR. The results were then compared with the clinical-pathological variables and outcome measures (Kaplan Meier and Cox analysis). Results: High expression of EGFR was observed in 43% of the samples and 20% of HPV detection. The statistical analyses provided evidence of disassociation between clinical-pathological parameters and EGFR expression, but there was an association with poor prognosis. Interestingly, HPV detection is slightly associated with good prognosis. Conclusion: Both, EGFR overexpression and HPV presence could be associated with an unfavorable prognosis in patients with LSCC, independently of other clinical-pathological factors.
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Carcinoma de Células Escamosas/mortalidad , Neoplasias Laríngeas/mortalidad , Infecciones por Papillomavirus/mortalidad , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , ADN Viral/genética , Receptores ErbB/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Laríngeas/patología , Neoplasias Laríngeas/virología , Masculino , México , Persona de Mediana Edad , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Pronóstico , Tasa de SupervivenciaRESUMEN
Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/fisiología , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Humanos , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Pinocitosis/fisiología , Ratas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-ß (TGF-ß) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle α-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-ß -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.
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Diferenciación Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Músculo Liso Vascular/citología , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Actinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Marcadores Genéticos , Humanos , Proteínas de Microfilamentos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fosfoproteínas/genética , Fosforilación , Transactivadores/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Quinasas Asociadas a rho/metabolismo , CalponinasRESUMEN
Nox generated ROS, particularly those derived from Nox1, Nox2 and Nox4, have emerged as important regulators of the actin cytoskeleton and cytoskeleton-supported cell functions, such as migration and adhesion. The effects of Nox-derived ROS on cytoskeletal remodeling may be largely attributed to the ability of ROS to directly modify proteins that constitute or are associated with the cytoskeleton. Additionally, Nox-derived ROS may participate in signaling pathways governing cytoskeletal remodeling. In addition to these more extensively studied signaling pathways involving Nox-derived ROS, there also exist redox sensitive pathways for which the source of ROS is unclear. ROS from as of yet undetermined sources play a role in modifying, and thus regulating, the activity of several proteins critical for remodeling of the actin cytoskeleton. In this review we discuss ROS sensitive targets that are likely to affect cytoskeletal dynamics, as well as the potential involvement of Nox proteins.
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Citoesqueleto/metabolismo , NADPH Oxidasas/metabolismo , Humanos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In 2010, in a cancer genes census, 291 genes were enumerated. These represent near to the 1 % of the total genes, for which there is enough biological evidence that they belong to a new genes classification, known as the cancer genes. These have been defined as the causal genes for sporadic or familiar cancer, when they mutate. The mutation types for these genes includes amplifications, point mutations, deletions, genomic rearranges, amongst others, which lead to a protein over-expression, muting, production of chimeric proteins or a de novo expression. In conjunction these genomic alterations or those of the genetic expression, when they affect specific genes which contribute to the development of cancer, are denominated as cancer genes. It is possible that the list of these alterations will grow longer due to new strategies being developed, for example, the genomic analysis.
En el año 2010, en un censo de genes del cáncer, se enumeraron 291 genes humanos que representan cerca del 1 % de los genes totales, para los cuales existe suficiente evidencia biológica de que pertenecen a una nueva clasificación de genes: los genes del cáncer. Estos se han definido como los genes causales de cáncer esporádico o cáncer familiar, cuando mutan. El tipo de mutaciones para estos genes del cáncer incluye las amplificaciones, las mutaciones puntuales, las deleciones, los rearreglos genómicos, entre otros, los cuales conducen a una sobreexpresión proteica, silenciamiento, producción de proteínas quiméricas o una expresión de novo. Cuando afectan genes específicos que contribuyen al desarrollo de un cáncer, estas alteraciones genómicas o de la expresión génica son denominadas en conjunto como genes del cáncer. Es posible que esta lista crezca más debido a las nuevas estrategias que se están desarrollando, como, por ejemplo, las de análisis genómico.
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Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Mutación , Neoplasias/genética , Genómica , HumanosRESUMEN
Cervical cancer (CC) is a multifactorial disease associated to genetic, environmental and epigenetic factors, being the infection by human papillomavirus the main etiologic agent. Additionally, the alteration in the expression of transcription factors has been considered of importance for the development of this tumor. HOX genes encode a group of transcription factors involved in cellular proliferation and differentiation processes during the development of embryonic structures in vertebrates; their aberrant expression is associated with tumorigenesis and metastasis. A range of evidence suggests a role for HOX genes in the development of cervical neoplastic cell. Studies in CC cell lines, primary tumors and premalignant lesions have suggested the involvement of HOXA1, HOXC5, C6, C8 and C10, HOXD9 and HOXD13 in the process of cervical carcinogenesis. Also, the de novo expression of genes HOXB2, B4, B13 and HOXC11-C13 appears to be involved in the process of malignant transformation of cervical epithelial cell. These data would allow to open a field in search of new molecular markers in cervical cancer and the development of new therapeutic strategies for this malignancy.
El cáncer cervicouterino (CaCU) es una enfermedad multifactorial que se asocia a factores genéticos, ambientales y epigenéticos, y cuyo principal agente etiológico es la infección por el virus del papiloma humano. Además, la alteración en la expresión de factores de transcripción ha sido considerada de importancia para el desarrollo de esta neoplasia. Los genes HOX codifican un grupo de factores de transcripción que participan en los procesos de proliferación y diferenciación celular durante el desarrollo de las estructuras embrionarias en los vertebrados; y su expresión aberrante ha sido asociada con oncogénesis y metástasis. Una serie de evidencias sugiere un papel importante para los genes HOX en el desarrollo neoplásico de la célula cervical. Estudios realizados en líneas celulares de CaCU, lesiones premalignas y tumores primarios han sugerido el involucramiento de HOXA1, HOXC5, C6, C8 y C10, HOXD9 y HOXD13 en el proceso de carcinogénesis cervical. Asimismo, la expresión de novo de los genes HOXB2, B4, B13 y HOXC11-C13 parece estar involucrada en el proceso de transformación maligna de la célula del epitelio cervical. Estos datos permitirían abrir un campo en la búsqueda de nuevos marcadores moleculares en cáncer cervical y en el desarrollo de nuevas estrategias terapéuticas para atender esta neoplasia.
Asunto(s)
Biomarcadores de Tumor/genética , Genes Homeobox , Neoplasias del Cuello Uterino/genética , Carcinogénesis/genética , Femenino , Marcadores Genéticos , Humanos , Metástasis de la Neoplasia/genéticaRESUMEN
Vascular smooth muscle cells (VSMCs) are subjected to various types of mechanical forces within the vessel wall. Although it is known that VSMCs undergo cell body reorientation in response to mechanical stimulation, how this mechanical stretch is transduced within the cell into biochemical signals causing cytoskeleton reorganization remains unclear. Cofilin, a protein that controls actin dynamics, is activated by Slingshot phosphatase-dependent serine 3 dephosphorylation by redox-dependent mechanisms. Nox4 is a main source of reactive oxygen species (ROS) in the vessel wall that localizes in association with the cytoskeleton. Therefore, we hypothesize that Nox4 mediates redox-dependent activation of cofilin, which is required for cytoskeletal reorganization and cell reorientation after mechanical stimulation. In this study, we found that mechanical stretch stimulates ROS production in VSMCs and that the signaling that leads to cell reorientation requires hydrogen peroxide but not superoxide. Indeed, mechanical stretch induces cofilin activation and stretch-induced cytoskeletal reorganization, and cell reorientation is inhibited in cells where cofilin activity has been downregulated. Importantly, Nox4-deficient cells fail to activate cofilin and to undergo cell reorientation, a phenotype rescued by the expression of a constitutively active cofilin mutant. Our results demonstrate that in VSMCs mechanical stimulation activates cofilin by a Nox4-dependent mechanism and that this pathway is required for cytoskeleton reorganization and cell reorientation.