RESUMO
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.
Assuntos
Glioblastoma , Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteína cdc42 de Ligação ao GTP , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Humanos , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Redes Reguladoras de Genes , Regulação Neoplásica da Expressão Gênica , Splicing de RNA , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Induced pluripotent stem cells (iPSCs) were successfully generated from peripheral blood mononuclear cells obtained from two patients with a heterozygous mutation in the CDC42 gene. Both iPSC lines expressed pluripotency markers, differentiated into the three germ layers in vitro, showed normal karyotypes, and retained the disease-causing mutation. Created iPSC lines and their differentiated derivatives may be of interest in the study of the physiology of disease mechanisms and therapy.
Assuntos
Diferenciação Celular , Heterozigoto , Células-Tronco Pluripotentes Induzidas , Mutação , Proteína cdc42 de Ligação ao GTP , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Masculino , FemininoRESUMO
Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.
Assuntos
Fatores de Despolimerização de Actina , Actinas , Espinhas Dendríticas , Hipocampo , Potenciação de Longa Duração , Animais , Espinhas Dendríticas/metabolismo , Potenciação de Longa Duração/fisiologia , Actinas/metabolismo , Hipocampo/metabolismo , Hipocampo/citologia , Fatores de Despolimerização de Actina/metabolismo , Ratos , Proteínas rac1 de Ligação ao GTP/metabolismo , Sinapses/metabolismo , Polimerização , Proteína cdc42 de Ligação ao GTP/metabolismo , Plasticidade Neuronal/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Neurônios/metabolismo , Transdução de Sinais , Camundongos , Células CultivadasRESUMO
To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.
Assuntos
Proteínas de Bactérias , Chlamydophila pneumoniae , Endocitose , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP , Chlamydophila pneumoniae/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Ligação Proteica , Nexinas de Classificação/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/genética , Interações Hospedeiro-Patógeno , Células HeLa , Membrana Celular/metabolismo , Mimetismo Molecular , Sítios de Ligação , Cristalografia por Raios XRESUMO
Emu oil is the oil extracted from the body fat of the Australian bird emu. Although previous studies have reported that emu oil has anti-inflammatory effects, the effect and mechanism of emu oil on the treatment of atopic dermatitis have not been reported. Here, 2, 4-dinitrofluorobenzene was used to induce atopic dermatitis-like appearance on the back skin of C57BL/6 mice. And then, the effect of emu oil in the atopic dermatitis treatment was evaluated. We found that emu oil reduced the transdermal water loss in the atopic dermatitis model. Additionally, the epidermal thickness treated with emu oil was significantly thinner. The number of mast cells and inflammatory cells were significantly decreased. The thymic stromal lymphopoietin (TSLP), which is secreted by keratinocyte, was decreased significantly after treatment. Moreover, the serum levels of cytokines TSLP, interleukin-4, interleukin-13, and immunoglobulin (Ig) E were decreased after emu oil treatment. Surprisingly, we found that the high level of Cdc42 expression in the atopic dermatitis, which was decreased after emu oil treatment. To detect the role of Cdc42 in atopic dermatitis, we constructed atopic dermatitis model in mice with sustained activation of Cdc42 signaling. Furthermore, we have confirmed that emu oil demonstrates anti-inflammatory effects in atopic dermatitis by inhibiting the expression of Cdc42 signaling in keratinocytes. In conclusion, we discovered a new role of Cdc42 in the development of atopic dermatitis, which mediated the therapeutic effect of emu oil on atopic dermatitis.
Assuntos
Anti-Inflamatórios , Citocinas , Dermatite Atópica , Modelos Animais de Doenças , Queratinócitos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP , Animais , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Citocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos , Proteína cdc42 de Ligação ao GTP/metabolismo , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/farmacologia , Linfopoietina do Estroma do Timo , Óleos/farmacologia , Óleos/uso terapêutico , Imunoglobulina E/sangue , Dinitrofluorbenzeno , Pele/efeitos dos fármacos , Pele/patologia , Pele/metabolismo , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , MasculinoRESUMO
The circular RNA (circRNA) family is a group of endogenous non-coding RNAs (ncRNAs) that have critical functions in multiple physiological and pathological processes, including inflammation, cancer, and cardiovascular diseases. However, their roles in regulating innate immune responses remain unclear. Here, we define Cell division cycle 42 (CDC42)-165aa, a protein encoded by circRNA circCDC42, which is overexpressed in Klebsiella pneumoniae (KP)-infected alveolar macrophages. High levels of CDC42-165aa induces the hyperactivation of Pyrin inflammasomes and aggravates alveolar macrophage pyroptosis, while the inhibition of CDC42-165aa reduces lung injury in mice after KP infection by inhibiting Pyrin inflammasome-mediated pyroptosis. Overall, these results demonstrate that CDC42-165aa stimulates Pyrin inflammasome by inhibiting CDC42 GTPase activation and provides a potential clinical target for pathogenic bacterial infection in clinical practice.
Assuntos
Inflamassomos , Infecções por Klebsiella , Klebsiella pneumoniae , Camundongos Endogâmicos C57BL , Piroptose , Proteína cdc42 de Ligação ao GTP , Animais , Piroptose/genética , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/metabolismo , Camundongos , Inflamassomos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Humanos , Imunidade Inata , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas Adaptadoras de Sinalização CARDRESUMO
Actin assembly and dynamics are crucial for maintaining cell structure and changing physiological states. The broad impact of actin on various cellular processes makes it challenging to dissect the specific role of actin regulatory proteins. Using actin waves that propagate on the cortex of mast cells as a model, we discovered that formins (FMNL1 and mDia3) are recruited before the Arp2/3 complex in actin waves. GTPase Cdc42 interactions drive FMNL1 oscillations, with active Cdc42 and the constitutively active mutant of FMNL1 capable of forming waves on the plasma membrane independently of actin waves. Additionally, the delayed recruitment of Arp2/3 antagonizes FMNL1 and active Cdc42. This antagonism is not due to competition for monomeric actin but rather for their common upstream regulator, active Cdc42, whose levels are negatively regulated by Arp2/3 via SHIP1 recruitment. Collectively, our study highlights the complex feedback loops in the dynamic control of the actin cytoskeletal network.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Forminas , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Forminas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Humanos , Mastócitos/metabolismo , Camundongos , Citoesqueleto de Actina/metabolismoRESUMO
The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Polaridade Celular , Endocitose , Retroalimentação Fisiológica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteína cdc42 de Ligação ao GTP , Quinases Ativadas por p21 , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Actinas/metabolismoRESUMO
Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.
Assuntos
Actinas , Desdiferenciação Celular , Condrócitos , Fibras de Estresse , Tropomiosina , Condrócitos/metabolismo , Condrócitos/citologia , Fibras de Estresse/metabolismo , Animais , Actinas/metabolismo , Tropomiosina/metabolismo , Tropomiosina/genética , Fenótipo , Células Cultivadas , Proteína cdc42 de Ligação ao GTP/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin.
Assuntos
Apigenina , Carcinoma Hepatocelular , Micropartículas Derivadas de Células , Neoplasias Hepáticas , Neovascularização Patológica , Fatores de Troca de Nucleotídeo Guanina Rho , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Animais , Apigenina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/irrigação sanguínea , Camundongos , Linhagem Celular Tumoral , Proteína cdc42 de Ligação ao GTP/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Hep G2 , Camundongos Nus , AngiogêneseRESUMO
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.
Assuntos
Diferenciação Celular , Disfunção Erétil , Células-Tronco Mesenquimais , MicroRNAs , Paxilina , RNA Longo não Codificante , Ratos Sprague-Dawley , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP , Quinases Ativadas por p21 , Masculino , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Ratos , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Células-Tronco Mesenquimais/metabolismo , Disfunção Erétil/terapia , Disfunção Erétil/genética , Disfunção Erétil/metabolismo , Paxilina/metabolismo , Paxilina/genética , Células Endoteliais/metabolismo , Células Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Aterosclerose , Movimento Celular , Células Espumosas , Camundongos Endogâmicos C57BL , Paxilina , Animais , Paxilina/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Camundongos , Aterosclerose/metabolismo , Aterosclerose/patologia , Transdução de Sinais , Lipoproteínas LDL/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Modelos Animais de Doenças , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Camundongos Knockout , Glicoproteínas de Membrana , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
5-Methylcytosine (m5C) methylation is a significant post-transcriptional modification that play a crucial role in the development and progression of numerous cancers. Whereas the functions and molecular mechanisms underlying m5C methylation in gliomas remain unclear. This study dedicated to explore changes of m5C levels and the clinical significance of the m5C writer NSUN4 in gliomas. We found that high m5C levels were negatively related to prognosis of patients with glioma. Moreover, gain- and loss-of-function experiments revealed the role of NSUN4 in enhancing m5C modification of mRNA to promote the malignant progression of glioma. Mechanistically speaking, NSUN4-mediated m5C alterations regulated ALYREF binding to CDC42 mRNA, thereby impacting the mRNA stability of CDC42. We also demonstrated that CDC42 promoted glioma proliferation, migration, and invasion by activating the PI3K-AKT pathway. Additionally, rescue experiments proved that CDC42 overexpression weaken the inhibitory effect of NSUN4 knockdown on the malignant progression of gliomas in vitro and in vivo. Our findings elucidated that NSUN4-mediated high m5C levels promote ALYREF binding to CDC42 mRNA and regulate its stability, thereby driving the malignant progression of glioma. This provides theoretical support for targeted the treatment of gliomas.
Assuntos
5-Metilcitosina , Glioma , Metiltransferases , Estabilidade de RNA , Proteína cdc42 de Ligação ao GTP , Animais , Feminino , Humanos , Masculino , Camundongos , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Glioma/metabolismo , Camundongos Nus , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
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.
Assuntos
Actinas , Actomiosina , Exoesqueleto , Gastrópodes , Morfogênese , Proteína cdc42 de Ligação ao GTP , Animais , Gastrópodes/embriologia , Gastrópodes/metabolismo , Exoesqueleto/metabolismo , Exoesqueleto/crescimento & desenvolvimento , Exoesqueleto/embriologia , Actinas/metabolismo , Actomiosina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Miosina Tipo II/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We previously reported trisubstituted pyrimidine lead compounds, namely, ARN22089 and ARN25062, which block the interaction between CDC42 with its specific downstream effector, a PAK protein. This interaction is crucial for the progression of multiple tumor types. Such inhibitors showed anticancer efficacy in vivo. Here, we describe a second class of CDC42 inhibitors with favorable drug-like properties. Out of the 25 compounds here reported, compound 15 (ARN25499) stands out as the best lead compound with an improved pharmacokinetic profile, increased bioavailability, and efficacy in an in vivo PDX tumor mouse model. Our results indicate that these CDC42 inhibitors represent a promising chemical class toward the discovery of anticancer drugs, with ARN25499 as an additional lead candidate for preclinical development.
Assuntos
Antineoplásicos , Proteína cdc42 de Ligação ao GTP , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/química , Humanos , Camundongos , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Pirimidinas/farmacocinética , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/síntese química , FemininoRESUMO
17ßestradiol (E2) can inhibit cardiac fibrosis in female patients with heart failure (HF) and activate cell division cycle 42 (Cdc42), however it is unknown whether 17ßestradiol (E2) can ameliorate differentiation and collagen synthesis in TGFß1stimulated mouse cardiac fibroblasts (MCFs) by regulating cell division cycle 42 (Cdc42). The present study aimed to investigate the roles of estrogen and Cdc42 in preventing myocardial fibrosis and the underlying molecular mechanisms. An ELISA was used to measure the levels of E2 and Cdc42 in the serum of patients with heart failure (HF), and western blotting was used to measure the expression levels of Cdc42 in TGFß1stimulated immortalized MCFs. MCFs were transfected with a Cdc42 overexpression (OE) lentivirus or small interfering RNA (siRNA), or treated with a Cdc42 inhibitor (MLS573151), and the function of Cdc42 was assessed by western blotting, immunofluorescence staining, reverse transcriptionquantitative PCR and dualluciferase reporter assays. Western blotting and immunofluorescence staining were performed to verify the protective effect of E2 on TGFß1stimulated MCFs, and the association between the protective effect and Cdc42. The results demonstrated that Cdc42 levels were increased in the serum of patients with HF and were positively correlated with the levels of E2; however, Cdc42 levels were decreased in TGFß1stimulated MCFs. Cdc42 inhibited MCF differentiation and collagen synthesis, as indicated by the protein expression of αsmooth muscle actin, collagen I and collagen III. Mechanistically, Cdc42 inhibited the transcription of TGFß1 by promoting the expression of p21 (RAC1)activated kinase 1 (Pak1)/JNK/cJun signaling pathway proteins and inhibiting the activity of the Tgfb1 gene promoter. In addition, E2 inhibited the differentiation and collagen synthesis of TGFß1stimulated MCFs, and promoted the protein expression of Pak1, JNK and cJun, consistent with the effects of Cdc42, whereas the effects of E2 were abolished when Cdc42 was knocked down. The aforementioned findings suggested that E2 could inhibit differentiation and collagen synthesis in TGFß1stimulated MCFs by regulating Cdc42 and the downstream Pak1/JNK/cJun signaling pathway.
Assuntos
Diferenciação Celular , Colágeno , Estradiol , Estrogênios , Fibroblastos , Fator de Crescimento Transformador beta1 , Proteína cdc42 de Ligação ao GTP , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Humanos , Colágeno/metabolismo , Colágeno/biossíntese , Feminino , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Estrogênios/farmacologia , Estradiol/farmacologia , Pessoa de Meia-Idade , Miocárdio/metabolismo , Insuficiência Cardíaca/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Astrócitos , Interleucina-10 , Metanfetamina , Camundongos Transgênicos , Microglia , Proteína cdc42 de Ligação ao GTP , Animais , Metanfetamina/toxicidade , Metanfetamina/farmacologia , Interleucina-10/metabolismo , Interleucina-10/farmacologia , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estimulantes do Sistema Nervoso Central/toxicidade , Estimulantes do Sistema Nervoso Central/farmacologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/induzido quimicamente , Células Cultivadas , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidadeRESUMO
BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.
Assuntos
Actinas , Meiose , Oócitos , Proteína cdc42 de Ligação ao GTP , Animais , Oócitos/metabolismo , Camundongos , Feminino , Actinas/metabolismo , Actinas/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Fosforilação , Fuso Acromático/metabolismoRESUMO
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.
Assuntos
Aminoquinolinas , Doxorrubicina , Células Endoteliais , Fibroblastos , Miócitos Cardíacos , Pirimidinas , Proteína cdc42 de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Animais , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Doxorrubicina/toxicidade , Doxorrubicina/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/metabolismo , Cardiotoxicidade , Antibióticos Antineoplásicos/toxicidade , Camundongos , Apoptose/efeitos dos fármacos , Masculino , Humanos , Camundongos Endogâmicos C57BL , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Neuropeptídeos/metabolismo , Dano ao DNA/efeitos dos fármacos , Células CultivadasRESUMO
Cell division cycle 42 (CDC42) regulates the progression of leukemia via mediating proliferation and immune evasion of malignant cells. The study aimed to investigate the correlation of CDC42 with clinical features, treatment response, event-free survival (EFS) and overall survival (OS) in adult Philadelphia chromosome negative acute lymphoblastic leukemia (Ph- ALL) patients. CDC42 expression in bone marrow mononuclear cells was detected in 78 adult Ph- ALL patients and 10 donors using real-time reverse transcriptase-polymerase chain reaction. CDC42 was increased in adult Ph- ALL patients compared with donors (p < .001). Besides, elevated CDC42 was linked with pro-B ALL or early-T ALL (p = .038) and white blood cell (WBC) elevation at diagnosis (p = .025). Fifty (64.1%) and 23 (29.5%) patients had complete remission (CR) at 1 month and minimal residual disease (MRD) after CR, respectively. CDC42 was inversely associated with CR at 1 month (p = .034), but not MRD after CR (p = .066). Concerning survival, patients with CDC42 ≥ 3.310 (cut by median value in patients) showed a shortened EFS (p = .006) and OS (p = .036) compared to those with CDC42 < 3.310. In detail, patients with CDC42 ≥ 3.310 and CDC42 < 3.310 had 5-year EFS rate of 29.9% and 45.4%, and 5-year OS rate of 39.4% and 63.6%, correspondingly. Further multivariate Cox's regression analyses revealed that CDC42 ≥ 3.310 was independently related to shorter EFS (hazard ratio = 2.933, p = .005). Elevated CDC42 is related with pro-B ALL or early-T ALL, WBC elevation at diagnosis, unfavorable treatment response and worse survival in adult Ph- ALL patients.