RESUMEN
Fibrosis can develop in most organs and causes organ failure. The most common type of lung fibrosis is known as idiopathic pulmonary fibrosis, in which fibrosis starts at the lung periphery and then progresses toward the lung center, eventually causing respiratory failure. Little is known about the mechanisms underlying the pathogenesis and periphery-to-center progression of the disease. Here we discovered that loss of Cdc42 function in alveolar stem cells (AT2 cells) causes periphery-to-center progressive lung fibrosis. We further show that Cdc42-null AT2 cells in both post-pneumonectomy and untreated aged mice cannot regenerate new alveoli, resulting in sustained exposure of AT2 cells to elevated mechanical tension. We demonstrate that elevated mechanical tension activates a TGF-ß signaling loop in AT2 cells, which drives the periphery-to-center progression of lung fibrosis. Our study establishes a direct mechanistic link between impaired alveolar regeneration, mechanical tension, and progressive lung fibrosis.
Asunto(s)
Células Madre Adultas/metabolismo , Fibrosis Pulmonar Idiopática/etiología , Alveolos Pulmonares/metabolismo , Células Madre Adultas/patología , Anciano , Células Epiteliales Alveolares/patología , Animales , Fenómenos Biomecánicos/fisiología , Femenino , Fibrosis/patología , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Alveolos Pulmonares/patología , Regeneración , Transducción de Señal , Células Madre/patología , Estrés Mecánico , Estrés Fisiológico/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.
Asunto(s)
Membrana Celular/química , Schizosaccharomyces/química , Schizosaccharomyces/citología , Actinas/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Microdominios de Membrana , Proteínas Asociadas a Microtúbulos/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Toll-like receptor (TLR) activation contributes to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS). TRAF6, a TLR effector with ubiquitin (Ub) ligase activity, is overexpressed in MDS hematopoietic stem/progenitor cells (HSPCs). We found that TRAF6 overexpression in mouse HSPC results in impaired hematopoiesis and bone marrow failure. Using a global Ub screen, we identified hnRNPA1, an RNA-binding protein and auxiliary splicing factor, as a substrate of TRAF6. TRAF6 ubiquitination of hnRNPA1 regulated alternative splicing of Arhgap1, which resulted in activation of the GTP-binding Rho family protein Cdc42 and accounted for hematopoietic defects in TRAF6-expressing HSPCs. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Síndromes Mielodisplásicos/inmunología , Lesiones Precancerosas/inmunología , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación , Animales , Autoinmunidad , Células Cultivadas , Hematopoyesis/genética , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/genética , Factor 6 Asociado a Receptor de TNF/genética , Receptores Toll-Like/metabolismo , Ubiquitinación/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.
Asunto(s)
Infecciones por Bacterias Gramnegativas/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemo/metabolismo , Hemólisis/inmunología , Macrófagos/inmunología , Fagocitosis , Sepsis/inmunología , Animales , Antibacterianos/uso terapéutico , Citoesqueleto/metabolismo , Femenino , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/genética , Hemo-Oxigenasa 1/genética , Hemólisis/efectos de los fármacos , Humanos , Evasión Inmune , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Quinina/uso terapéutico , Células RAW 264.7 , Sepsis/tratamiento farmacológico , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.
Asunto(s)
Neoplasias de la Próstata , Proteínas de Unión al GTP rho , Humanos , Masculino , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFß pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.
Asunto(s)
Células-Madre Neurales , Proteínas de Unión al GTP rho , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Diferenciación Celular , Transducción de Señal , Neurogénesis , Células-Madre Neurales/metabolismoRESUMEN
In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.
Asunto(s)
Actinas , Actomiosina , Exoesqueleto , Gastrópodos , Morfogénesis , Proteína de Unión al GTP cdc42 , Animales , Gastrópodos/embriología , Gastrópodos/metabolismo , Exoesqueleto/metabolismo , Exoesqueleto/crecimiento & desarrollo , Exoesqueleto/embriología , Actinas/metabolismo , Actomiosina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Miosina Tipo II/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Increasing evidence highlights the role of bacteria in promoting tumorigenesis. The underlying mechanisms may be diverse and remain poorly understood. Here, we report that Salmonella infection leads to extensive de/acetylation changes in host cell proteins. The acetylation of mammalian cell division cycle 42 (CDC42), a member of the Rho family of GTPases involved in many crucial signaling pathways in cancer cells, is drastically reduced after bacterial infection. CDC42 is deacetylated by SIRT2 and acetylated by p300/CBP. Non-acetylated CDC42 at lysine 153 shows an impaired binding of its downstream effector PAK4 and an attenuated phosphorylation of p38 and JNK, consequently reduces cell apoptosis. The reduction in K153 acetylation also enhances the migration and invasion ability of colon cancer cells. The low level of K153 acetylation in patients with colorectal cancer (CRC) predicts a poor prognosis. Taken together, our findings suggest a new mechanism of bacterial infection-induced promotion of colorectal tumorigenesis by modulation of the CDC42-PAK axis through manipulation of CDC42 acetylation.
Asunto(s)
Neoplasias Colorrectales , Infecciones por Salmonella , Proteína de Unión al GTP cdc42 , Humanos , Acetilación , Carcinogénesis , Proteína de Unión al GTP cdc42/metabolismo , Transformación Celular Neoplásica , Quinasas p21 Activadas/metabolismo , Transducción de SeñalRESUMEN
Synapses change their weights in response to neuronal activity and in turn, neuronal networks alter their response properties and ultimately allow the brain to store information as memories. As for memories, not all events are maintained over time. Maintenance of synaptic plasticity depends on the interplay between functional changes at synapses and the synthesis of plasticity-related proteins that are involved in stabilizing the initial functional changes. Different forms of synaptic plasticity coexist in time and across the neuronal dendritic area. Thus, homosynaptic plasticity refers to activity-dependent synaptic modifications that are input-specific, whereas heterosynaptic plasticity relates to changes in non-activated synapses. Heterosynaptic forms of plasticity, such as synaptic cooperation and competition allow neurons to integrate events that occur separated by relatively large time windows, up to one hour. Here, we show that activation of Cdc42, a Rho GTPase that regulates actin cytoskeleton dynamics, is necessary for the maintenance of long-term potentiation (LTP) in a time-dependent manner. Inhibiting Cdc42 activation does not alter the time-course of LTP induction and its initial expression but blocks its late maintenance. We show that Cdc42 activation is involved in the phosphorylation of cofilin, a protein involved in modulating actin filaments and that weak and strong synaptic activation leads to similar levels on cofilin phosphorylation, despite different levels of LTP expression. We show that Cdc42 activation is required for synapses to interact by cooperation or competition, supporting the hypothesis that modulation of the actin cytoskeleton provides an activity-dependent and time-restricted permissive state of synapses allowing synaptic plasticity to occur. We found that under competition, the sequence in which synapses are activated determines the degree of LTP destabilization, demonstrating that competition is an active destabilization process. Taken together, we show that modulation of actin cytoskeleton by Cdc42 activation is necessary for the expression of homosynaptic and heterosynaptic forms of plasticity. Determining the temporal and spatial rules that determine whether synapses cooperate or compete will allow us to understand how memories are associated.
Asunto(s)
Potenciación a Largo Plazo , Sinapsis , Proteína de Unión al GTP cdc42 , Proteína de Unión al GTP cdc42/metabolismo , Animales , Potenciación a Largo Plazo/fisiología , Sinapsis/metabolismo , Sinapsis/fisiología , Fosforilación , Plasticidad Neuronal/fisiología , Ratas , Hipocampo/metabolismo , Hipocampo/fisiología , Hipocampo/citología , Factores Despolimerizantes de la Actina/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , MasculinoRESUMEN
Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.
Asunto(s)
Movimiento Celular , Kisspeptinas , Miocitos del Músculo Liso , Factor de Crecimiento Derivado de Plaquetas , Receptores de Kisspeptina-1 , Transducción de Señal , Proteína de Unión al GTP rhoA , Humanos , Movimiento Celular/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Kisspeptinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Kisspeptina-1/metabolismo , Receptores de Kisspeptina-1/genética , Proteína de Unión al GTP rhoA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Remodelación de las Vías Aéreas (Respiratorias) , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Cultivadas , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Proliferación CelularRESUMEN
The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.
Asunto(s)
Proteína de Unión al GTP cdc42 , Proteínas de Unión al GTP rho , Animales , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Polaridad Celular/fisiología , Proteínas de Unión al GTP rho/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
Asunto(s)
Proteínas del Citoesqueleto , Salmonella typhimurium , Proteínas de Unión al GTP rho , Humanos , Actinas/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Citoesqueleto/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Salmonella typhimurium/patogenicidad , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismoRESUMEN
Methamphetamine (Meth) use is known to induce complex neuroinflammatory responses, particularly involving astrocytes and microglia. Building upon our previous research, which demonstrated that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, this study investigates the role of the anti-inflammatory cytokine interleukin-10 (IL-10) in this process. Our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocyte cultures, which significantly reduces microglial activation. This reduction is associated with the modulation of astrocytic intracellular calcium (Ca2+) dynamics, particularly by restricting the release of Ca2+ from the endoplasmic reticulum to the cytoplasm. Furthermore, we identify the small Rho GTPase Cdc42 as a crucial intermediary in the astrocyte-to-microglia communication pathway under Meth exposure. By employing a transgenic mouse model that overexpresses IL-10 (pMT-10), we also demonstrate in vivo that IL-10 prevents Meth-induced neuroinflammation. These findings not only enhance our understanding of Meth-related neuroinflammatory mechanisms, but also suggest IL-10 and Cdc42 as putative therapeutic targets for treating Meth-induced neuroinflammation.
Asunto(s)
Astrocitos , Interleucina-10 , Metanfetamina , Ratones Transgénicos , Microglía , Proteína de Unión al GTP cdc42 , Animales , Metanfetamina/toxicidad , Metanfetamina/farmacología , Interleucina-10/metabolismo , Interleucina-10/farmacología , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Proteína de Unión al GTP cdc42/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Ratones , Ratones Endogámicos C57BL , Estimulantes del Sistema Nervioso Central/toxicidad , Estimulantes del Sistema Nervioso Central/farmacología , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/inducido químicamente , Células Cultivadas , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidadRESUMEN
Gene co-expression networks may encode hitherto inadequately recognized vulnerabilities for adult gliomas. By identifying evolutionally conserved gene co-expression modules around EGFR (EM) or PDGFRA (PM), we recently proposed an EM/PM classification scheme, which assigns IDH-wildtype glioblastomas (GBM) into the EM subtype committed in neural stem cell compartment, IDH-mutant astrocytomas and oligodendrogliomas into the PM subtype committed in early oligodendrocyte lineage. Here, we report the identification of EM/PM subtype-specific gene co-expression networks and the characterization of hub gene polypyrimidine tract-binding protein 1 (PTBP1) as a genomic alteration-independent vulnerability in IDH-wildtype GBM. Supervised by the EM/PM classification scheme, we applied weighted gene co-expression network analysis to identify subtype-specific global gene co-expression modules. These gene co-expression modules were characterized for their clinical relevance, cellular origin and conserved expression pattern during brain development. Using lentiviral vector-mediated constitutive or inducible knockdown, we characterized the effects of PTBP1 on the survival of IDH-wildtype GBM cells, which was complemented with the analysis of PTBP1-depedent splicing pattern and overexpression of splicing target neuron-specific CDC42 (CDC42-N) isoform. Transcriptomes of adult gliomas can be robustly assigned into 4 large gene co-expression modules that are prognostically relevant and are derived from either malignant cells of the EM/PM subtypes or tumor microenvironment. The EM subtype is associated with a malignant cell-intrinsic gene module involved in pre-mRNA splicing, DNA replication and damage response, and chromosome segregation, and a microenvironment-derived gene module predominantly involved in extracellular matrix organization and infiltrating immune cells. The PM subtype is associated with two malignant cell-intrinsic gene modules predominantly involved in transcriptional regulation and mRNA translation, respectively. Expression levels of these gene modules are independent prognostic factors and malignant cell-intrinsic gene modules are conserved during brain development. Focusing on the EM subtype, we identified PTBP1 as the most significant hub for the malignant cell-intrinsic gene module. PTBP1 is not altered in most glioma genomes. PTBP1 represses the conserved splicing of CDC42-N. PTBP1 knockdown or CDC42-N overexpression disrupts actin cytoskeleton dynamics, causing accumulation of reactive oxygen species and cell apoptosis. PTBP1-mediated repression of CDC42-N splicing represents a potential genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype GBM.
Asunto(s)
Glioblastoma , Ribonucleoproteínas Nucleares Heterogéneas , Proteína de Unión al Tracto de Polipirimidina , Proteína de Unión al GTP cdc42 , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Humanos , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Línea Celular Tumoral , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Redes Reguladoras de Genes , Regulación Neoplásica de la Expresión Génica , Empalme del ARN , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
Attainment of proper cell shape and the regulation of cell migration are essential processes in the development of an organism. The mixed lineage leukemia (MLL or KMT2A) protein, a histone 3 lysine 4 (H3K4) methyltransferase, plays a critical role in cell-fate decisions during skeletal development and haematopoiesis in higher vertebrates. Rho GTPases - RhoA, Rac1 and CDC42 - are small G proteins that regulate various key cellular processes, such as actin cytoskeleton formation, the maintenance of cell shape and cell migration. Here, we report that MLL regulates the homeostasis of these small Rho GTPases. Loss of MLL resulted in an abnormal cell shape and a disrupted actin cytoskeleton, which lead to diminished cell spreading and migration. MLL depletion affected the stability and activity of Rho GTPases in a SET domain-dependent manner, but these Rho GTPases were not direct transcriptional targets of MLL. Instead, MLL regulated the transcript levels of their chaperone protein RhoGDI1 (also known as ARHGDIA). Using MDA-MB-231, a triple-negative breast cancer cell line with high RhoGDI1 expression, we show that MLL depletion or inhibition by small molecules reduces tumour progression in nude mice. Our studies highlight the central regulatory role of MLL in Rho/Rac/CDC42 signalling pathways. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Unión al GTP rho , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Ratones , Animales , Inhibidor alfa de Disociación del Nucleótido Guanina rho/genética , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Ratones Desnudos , Histonas/metabolismo , Lisina , Transducción de Señal/fisiología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Movimiento Celular/fisiología , Citoesqueleto de Actina/metabolismo , Metiltransferasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Actinas/metabolismoRESUMEN
BACKGROUND: Macrophage-derived foam cell formation is a hallmark of atherosclerosis and is retained during plaque formation. Strategies to inhibit the accumulation of these cells hold promise as viable options for treating atherosclerosis. Plexin D1 (PLXND1), a member of the Plexin family, has elevated expression in atherosclerotic plaques and correlates with cell migration; however, its role in macrophages remains unclear. We hypothesize that the guidance receptor PLXND1 negatively regulating macrophage mobility to promote the progression of atherosclerosis. METHODS: We utilized a mouse model of atherosclerosis based on a high-fat diet and an ox-LDL- induced foam cell model to assess PLXND1 levels and their impact on cell migration. Through western blotting, Transwell assays, and immunofluorescence staining, we explored the potential mechanism by which PLXND1 mediates foam cell motility in atherosclerosis. RESULTS: Our study identifies a critical role for PLXND1 in atherosclerosis plaques and in a low-migration capacity foam cell model induced by ox-LDL. In the aortic sinus plaques of ApoE-/- mice, immunofluorescence staining revealed significant upregulation of PLXND1 and Sema3E, with colocalization in macrophages. In macrophages treated with ox-LDL, increased expression of PLXND1 led to reduced pseudopodia formation and decreased migratory capacity. PLXND1 is involved in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK. Additionally, FAK inhibitors counteract the ox-LDL-induced migration suppression by modulating the phosphorylation states of FAK, Paxillin and their downstream effectors CDC42 and PAK. CONCLUSION: Our findings indicate that PLXND1 plays a role in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK to promoting atherosclerosis.
Asunto(s)
Aterosclerosis , Movimiento Celular , Células Espumosas , Ratones Endogámicos C57BL , Paxillin , Animales , Paxillin/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patología , Ratones , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transducción de Señal , Lipoproteínas LDL/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteína de Unión al GTP cdc42/metabolismo , Macrófagos/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Modelos Animales de Enfermedad , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Ratones Noqueados , Glicoproteínas de Membrana , Péptidos y Proteínas de Señalización IntracelularRESUMEN
The control of size and shape is an important part of regulatory process during organogenesis. Tooth formation is a highly complex process that fine-tunes the size and shape of the tooth, which are crucial for its physiological functions. Each tooth consists of a crown and one or more roots. Despite comprehensive knowledge of the mechanism that regulates early tooth crown development, we have limited understanding of the mechanism regulating root patterning and size during development. Here, we show that Ror2-mediated non-canonical Wnt signaling in the dental mesenchyme plays a crucial role in cell proliferation, and thereby regulates root development size in mouse molars. Furthermore, Cdc42 acts as a potential downstream mediator of Ror2 signaling in root formation. Importantly, activation of Cdc42 can restore cell proliferation and partially rescue the root development size defects in Ror2 mutant mice. Collectively, our findings provide novel insights into the function of Ror2-mediated non-canonical Wnt signaling in regulating tooth morphogenesis, and suggest potential avenues for dental tissue engineering.
Asunto(s)
Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Raíz del Diente/embriología , Raíz del Diente/metabolismo , Vía de Señalización Wnt , Proteína de Unión al GTP cdc42/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Femenino , Masculino , Mesodermo/embriología , Ratones , Ratones Mutantes , Morfogénesis , Odontoblastos/citología , Odontoblastos/metabolismo , Raíz del Diente/citologíaRESUMEN
BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRTâPCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male SpragueâDawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRTâPCR, Western blot analysis, and immunohistochemical staining. RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups. CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.
Asunto(s)
Diferenciación Celular , Disfunción Eréctil , Células Madre Mesenquimatosas , MicroARNs , Paxillin , ARN Largo no Codificante , Ratas Sprague-Dawley , Transducción de Señal , Proteína de Unión al GTP cdc42 , Quinasas p21 Activadas , Masculino , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/genética , Ratas , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Disfunción Eréctil/terapia , Disfunción Eréctil/genética , Disfunción Eréctil/metabolismo , Paxillin/metabolismo , Paxillin/genética , Células Endoteliales/metabolismo , Células Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.
Asunto(s)
Aminoquinolinas , Doxorrubicina , Células Endoteliales , Fibroblastos , Miocitos Cardíacos , Pirimidinas , Proteína de Unión al GTP cdc42 , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína de Unión al GTP cdc42/metabolismo , Doxorrubicina/toxicidad , Doxorrubicina/efectos adversos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Células Endoteliales/metabolismo , Cardiotoxicidad , Antibióticos Antineoplásicos/toxicidad , Ratones , Apoptosis/efectos de los fármacos , Masculino , Humanos , Ratones Endogámicos C57BL , Roturas del ADN de Doble Cadena/efectos de los fármacos , Neuropéptidos/metabolismo , Daño del ADN/efectos de los fármacos , Células CultivadasRESUMEN
Ubiquitination serves as a critical signal in the host immune response to infection. Many pathogens have evolved strategies to exploit the ubiquitin (Ub) system to promote their own survival through a complex interplay between host defense machinery and bacterial virulence factors. Here we report dynamic changes in the global ubiquitinome of host epithelial cells and invading pathogen in response to Salmonella Typhimurium infection. The most significant alterations in the host ubiquitinome concern components of the actin cytoskeleton, NF-κB and autophagy pathways, and the Ub and RHO GTPase systems. Specifically, infection-induced ubiquitination promotes CDC42 activity and linear ubiquitin chain formation, both being required for NF-κB activation. Conversely, the bacterial ubiquitinome exhibited extensive ubiquitination of various effectors and several outer membrane proteins. Moreover, we reveal that bacterial Ub-modifying enzymes modulate a unique subset of host targets, affecting different stages of Salmonella infection.