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1.
Proc Natl Acad Sci U S A ; 120(43): e2303794120, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37844230

RESUMO

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.


Assuntos
Arrestinas , Transdução de Sinais , beta-Arrestinas/metabolismo , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Arrestinas/metabolismo , Regulação Alostérica , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Fosforilação , beta-Arrestina 2/metabolismo
2.
J Biol Chem ; 300(5): 107253, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38569938

RESUMO

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which in turn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later finding demonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.


Assuntos
Homocisteína , Mitocôndrias , Espécies Reativas de Oxigênio , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Homocisteína/metabolismo , Homocisteína/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Mitocôndrias/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Cálcio/metabolismo , Fosforilação/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Quinases da Família src/metabolismo , Ratos , Camundongos , Humanos
3.
Mol Microbiol ; 120(5): 684-701, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37718557

RESUMO

Syphilis is a persistent sexually transmitted disease caused by infiltration of the elusive pathogen Treponema pallidum. Despite the prevalence of human polymorphonuclear neutrophils (hPMNs) within cutaneous lesions, which are characteristic of incipient syphilis, their role in T. pallidum infection remains unclear. Tp92 is the only T. pallidum helical outer membrane protein that exhibits structural features similar to those of outer membrane proteins in other gram-negative bacteria. However, the functional mechanism of this protein in immune cells remains unclear. Neutrophils are short-lived cells that undergo innate apoptosis in response to external stimuli that typically influence this process. In this study, we determined that Tp92 impedes the activation of procaspase-3 via the ERK MAPK, PI3K/Akt, and NF-κB signaling pathways, consequently suppressing caspase-3 activity within hPMNs, and thereby preventing hPMNs apoptosis. Furthermore, Tp92 could also modulate hPMNs apoptosis by enhancing the expression of the anti-apoptotic protein Mcl-1, stimulating IL-8 secretion, and preserving the mitochondrial membrane potential. These findings provide valuable insights into the molecular mechanisms underlying T. pallidum infection and suggest potential therapeutic targets for syphilis treatment.


Assuntos
NF-kappa B , Sífilis , Humanos , NF-kappa B/metabolismo , Treponema pallidum/genética , Treponema pallidum/metabolismo , Sífilis/metabolismo , Sífilis/microbiologia , Sífilis/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos , Apoptose
4.
J Neuroinflammation ; 21(1): 271, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39438980

RESUMO

Hypertension is associated with poor outcome and higher mortality in patients with ischemic stroke. The impairment of adaptive vascular mechanisms under hypertensive condition compromises collateral blood flow after arterial occlusion in patients with acute ischemic stroke resulting in hypoperfusion. The increased oxidative stress caused by hypoperfusion is thought to be a trigger for the rapid evolution of ischemic infarct volume under hypertensive condition. However, the cellular factors and pathways that contribute to the exacerbation of ischemic brain injury under hypertensive condition is not yet understood. The current study reveals that predisposition to hypertension leads to basal loss of function of the neuron-specific tyrosine phosphatase STEP, which plays a crucial role in neuroprotection against excitotoxic insult. The findings further show that a mild ischemic insult in hypertensive rats triggers an early onset and sustained activation of the neuronal extracellular signal regulated kinase (ERK MAPK), a member of the mitogen activated protein kinase family and a substrate of STEP. This leads to rapid increase in the activation of neuronal NF-κB, expression of neuronal cyclooxygenase-2 and subsequent biosynthesis of the pro-inflammatory mediator prostaglandin E2, resulting in rapid morphological transformation of microglia to the pro-inflammatory state and subsequent exacerbation of ischemic brain injury. Restoration of STEP signaling with intravenous administration of a STEP-derived peptide mimetic reduces the pro-inflammatory response in neurons, activation of microglia, and ischemic brain injury. The findings suggest that the basal loss of STEP function under hypertensive condition contributes to the exacerbation of ischemic brain injury by enhancing post-ischemic inflammatory response. The study not only presents a novel role of STEP in regulating neuroimmune communication but also highlights the therapeutic potential of a STEP-mimetic in mitigating ischemic brain damage under hypertensive condition.


Assuntos
Hipertensão , Animais , Ratos , Hipertensão/patologia , Hipertensão/complicações , Masculino , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ratos Sprague-Dawley , Lesões Encefálicas/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/etiologia , Ratos Endogâmicos SHR , Isquemia Encefálica/patologia , Isquemia Encefálica/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos
5.
Genes Cells ; 28(6): 457-465, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36945130

RESUMO

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Neoplasias Pancreáticas , Humanos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Transdução de Sinais , Neoplasias Pancreáticas/tratamento farmacológico , Apoptose , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas
6.
Arch Biochem Biophys ; 754: 109896, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38417691

RESUMO

AIMS: The purpose of this study was to explore the role of RAE1 in the invasion and metastasis of gastric cancer (GC) cells. MATERIALS AND METHODS: RAE1 expression in GC cells was determined by reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting (WB). Cell models featuring RAE1 gene silencing and overexpression were constructed by lentiviral transfection; The proliferation, migration, and invasion ability of cells were detected by cell counting, colony formation assay, would healing assay, and transwell invasion and migration test. WB analysis of ERK/MAPK signaling pathway (ERK1/2, p-ERK1/2, c-Myc) and EMT-related molecules (ZEB1, E-cadherin, N-cadherin, and Vimentin). RESULTS: The expression level of RAE1 in GC was notably higher than in adjacent tissues. Elevated RAE1 expression correlated with an unfavorable prognosis for GC patients. Knockdown of RAE1, as compared to the control group, resulted in a significant inhibition of proliferation, migration, and invasion abilities in GC cell lines. Furthermore, RAE1 knockdown led to a substantial decrease in the expression of N-cadherin, vimentin, ZEB1, p-ERK1/2, and c-Myc proteins, coupled with a marked increase in E-cadherin expression. The biological effects of RAE1 in GC cells were effectively reversed by the inhibition of the ERK/MAPK signaling pathway using SCH772984. Additionally, RAE1 knockdown demonstrated a suppressive effect on GC tumor size in vivo. Immunohistochemistry (IHC) results revealed significantly lower expression of Ki-67 in RAE1 knockout mice compared to the control group. CONCLUSIONS: RAE1 promotes GC cell migration and invasion through the ERK/MAPK pathway and is a potential therapeutic target for GC therapy.


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Gástricas , Animais , Humanos , Camundongos , Caderinas/genética , Caderinas/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Vimentina/genética , Vimentina/metabolismo
7.
Metab Brain Dis ; 39(3): 387-401, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37284987

RESUMO

Autism Spectrum Disorders (ASD) are a complex set of neurodevelopmental manifestations which present in the form of social and communication deficits. Affecting a growing proportion of children worldwide, the exact pathogenesis of this disorder is not very well understood, and multiple signaling pathways have been implicated. Among them, the ERK/MAPK pathway is critical in a number of cellular processes, and the normal functioning of neuronal cells also depends on this cascade. As such, recent studies have increasingly focused on the impact this pathway has on the development of autistic symptoms. Improper ERK signaling is suspected to be involved in neurotoxicity, and the same might be implicated in autism spectrum disorders (ASD), through a variety of effects including mitochondrial dysfunction and oxidative stress. Niclosamide, an antihelminthic and anti-inflammatory agent, has shown potential in inhibiting this pathway, and countering the effects shown by its overactivity in inflammation. While it has previously been evaluated in other neurological disorders like Alzheimer's Disease and Parkinson's Disease, as well as various cancers by targeting ERK/MAPK, it's efficacy in autism has not yet been evaluated. In this article, we attempt to discuss the potential role of the ERK/MAPK pathway in the pathogenesis of ASD, specifically through mitochondrial damage, before moving to the therapeutic potential of niclosamide in the disorder, mediated by the inhibition of this pathway and its detrimental effects of neuronal development.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Doenças Mitocondriais , Criança , Humanos , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/metabolismo , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Estresse Oxidativo
8.
Acta Med Okayama ; 78(1): 1-8, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38419308

RESUMO

Liver fibrosis, which ultimately leads to liver cirrhosis and hepatocellular carcinoma, is a major health burden worldwide. The progression of liver fibrosis is the result of the wound-healing response of liver to repeated injury. Hepatic macrophages are cells with high heterogeneity and plasticity and include tissue-resident macrophages termed Kupffer cells, and recruited macrophages derived from circulating monocytes, spleen and peritoneal cavity. Studies have shown that hepatic macrophages play roles in the initiation and progression of liver fibrosis by releasing inflammatory cytokines/chemokines and pro-fibrogenic factors. Furthermore, the development of liver fibrosis has been shown to be reversible. Hepatic macrophages have been shown to alternately regulate both the regression and turnover of liver fibrosis by changing their phenotypes during the dynamic progression of liver fibrosis. In this review, we summarize the role of hepatic macrophages in the progression and regression of liver fibrosis.


Assuntos
Cirrose Hepática , Neoplasias Hepáticas , Humanos , Cirrose Hepática/patologia , Macrófagos/patologia , Fígado/lesões , Células de Kupffer/patologia , Neoplasias Hepáticas/patologia , Fibrose
9.
Int J Mol Sci ; 25(20)2024 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-39456824

RESUMO

Growth-factor-induced cell signaling plays a crucial role in development; however, negative regulation of this signaling pathway is important for sustaining homeostasis and preventing diseases. SPROUTY2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase (RTK) signaling that binds to GRB2 during RTK activation and inhibits the GRB2-SOS complex, which inhibits RAS activation and attenuates the downstream RAS/ERK signaling cascade. SPRY was formerly discovered in Drosophila but was later discovered in higher eukaryotes and was found to be connected to many developmental abnormalities. In several experimental scenarios, increased SPRY2 protein levels have been observed to be involved in both peripheral and central nervous system neuronal regeneration and degeneration. SPRY2 is a desirable pharmaceutical target for improving intracellular signaling activity, particularly in the RAS/ERK pathway, in targeted cells because of its increased expression under pathological conditions. However, the role of SPRY2 in brain-derived neurotrophic factor (BDNF) signaling, a major signaling pathway involved in nervous system development, has not been well studied yet. Recent research using a variety of small-animal models suggests that SPRY2 has substantial therapeutic promise for treating a range of neurological conditions. This is explained by its function as an intracellular ERK signaling pathway inhibitor, which is connected to a variety of neuronal activities. By modifying this route, SPRY2 may open the door to novel therapeutic approaches for these difficult-to-treat illnesses. This review integrates an in-depth analysis of the structure of SPRY2, the role of its major interactive partners in RTK signaling cascades, and their possible mechanisms of action. Furthermore, this review highlights the possible role of SPRY2 in neurodevelopmental disorders, as well as its future therapeutic implications.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Transtornos do Neurodesenvolvimento , Transdução de Sinais , Humanos , Animais , Transtornos do Neurodesenvolvimento/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Retroalimentação Fisiológica , Sistema de Sinalização das MAP Quinases
10.
J Biol Chem ; 298(12): 102661, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334633

RESUMO

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.


Assuntos
Genes ras , Transdução de Sinais , Humanos , Ligação Proteica , Transdução de Sinais/genética , Mutação , Guanosina Trifosfato/genética
11.
Biol Reprod ; 109(4): 533-551, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37552049

RESUMO

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.


Assuntos
Células-Tronco Adultas , MAP Quinases Reguladas por Sinal Extracelular , Masculino , Camundongos , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação , Diferenciação Celular/fisiologia , Espermatogônias/metabolismo , Células-Tronco Adultas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mamíferos/metabolismo
12.
J Transl Med ; 21(1): 766, 2023 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-37904179

RESUMO

BACKGROUND: Gastric cancer (GC) ranks as the fifth most common cancer worldwide and is characterized by its significant heterogeneity and unfavorable prognosis. Thus, identifying efficient prognostic factors and understanding the underlying molecular mechanisms in GC are essential for improving patient outcomes. In this study, we aimed to investigate the role of RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the prognostic significance and molecular mechanisms of its biological function in GC. METHODS: Multiple bioinformatics strategies were performed to detect the potential functions and prognostic efficiency of RECK in GC. Rescue experiments revealed that the molecular mechanism by which RECK in inhibited tumor proliferation, migration, and invasion was mediated by ERK/MAPK signaling in AGS and HGC-27 cells. Using integrated bioinformatics analysis and western blot assay, we investigated the potential interaction between CALD1 and RECK. RESULTS: Our findings revealed significantly decreased RECK expression in GC samples compared to normal samples and RECK was identified as a promising predictor for the prognosis of GC patients. Moreover, upregulation of RECK demonstrated a distinctly positive association with a high-immunity and low-metastasis microenvironment in GC. Mechanistically, the antitumour effects of RECK on hampering tumor cell growth, migration, and invasion were mediated by the ERK/MAPK signaling pathway. In addition, we also illustrated that RECK inhibited the phosphorylation of CALD1 mediated by decreased phosphorylation of ERK. CONCLUSIONS: RECK is a promising prognostic biomarker and may shape a high-tumor-immunity and low-metastasis microenvironment in patients with GC. Moreover, RECK exerted its tumor-suppressive effects by the inactivation of ERK/MAPK signaling in GC cells.


Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/metabolismo , Prognóstico , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/metabolismo , Transdução de Sinais , Regulação Neoplásica da Expressão Gênica , Microambiente Tumoral
13.
Biomarkers ; 28(3): 289-301, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36588463

RESUMO

Background: Carpet dust exposure in the carpet industry causes various respiratory hazards that lead to permanent loss of lung function. This study investigated the potentially toxic effects of knotted and tufted carpet dust on rat lungs and the possible involvement of cytochrome P450 2E1 (CYP2E1) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways in the induced toxicity, as well as histological changes in the lung induced by carpet dust.Methods: This study divided 48 adult rats into six groups: group I was the control group, group II (vehicle group) received phosphate buffer saline (50 µL/rat), groups III and IV received knotted dust (2.5 and 5 mg/kg, respectively), and groups V and VI received tufted dust (2.5 and 5 mg/kg, respectively). All treatments were intranasally administered once a day for 7 days.Results: Both dust types significantly decreased the lung content of GSH compared with the control. Significantly elevated malondialdehyde (MDA) and nitric oxide (NO) lung contents were observed with an increased CYP2E1, interleukin (IL)-6, nuclear factor kappa B (NF-κß), and ERK/MAPK. The histological lung structure was moderately affected with a moderately increased number of CD68-positive macrophages in the lung parenchyma of knotted dust-exposed rats, whereas tufted dust exposure severely affected the lung tissue with significantly increased CD68-positive macrophages.Conclusions: Carpet dust exposure could induce oxidative stress and inflammatory response in the lung tissue via induction of CYP2E1 that stimulates ERK/MAPK signalling pathway proteins, resulting in elevated MDA, NO and IL-6 levels in the lung tissue with suppressed GSH content. Tufted dust could possess a more toxic response than knotted ones.


Assuntos
Citocromo P-450 CYP2E1 , Poeira , Ratos , Animais , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP2E1/farmacologia , Pisos e Cobertura de Pisos , Pulmão/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/farmacologia
14.
Exp Cell Res ; 410(1): 112956, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34864005

RESUMO

The ERK/MAPK signaling pathway is activated in various cancers including gastric cancer. Targeting the ERK/MAPK/MEK pathway has been considered as a promising strategy for cancer therapy. However, MEK inhibition leads to a series of resistance mechanisms due to mutations in MEK, elevated expression of RAS or RAF proteins and activation of the associated signaling pathways. In the present study, ERK/MAPK pathway specific gene signatures were identified to be highly activated in intestinal subtype gastric tumors. Inhibition of ERK/MAPK pathway with the inhibitor PD98059 in gastric cancer cell lines by in vitro signaling pathway and genome-wide expression profiling revealed the associated signaling pathways. Functional genomic investigation of the ERK/MAPK regulated genes reveals the association of ERK/MAPK pathway with E2F, Myc, SOX-2, TGF-ß, OCT4 and Notch pathways in gastric cancer cells. Of these, E2F, Myc and SOX-2 pathways are activated in intestinal subtype gastric tumors and TGF-ß, OCT4, Notch pathways are activated in diffuse subtype gastric tumors. Further, the mutational load of gastric tumors was found to have association and correlation with the activation pattern of ERK/MAPK pathways across gastric tumors. ERK/MAPK activation was also found to represent the EBV and MSI activated subtypes of gastric tumors. Identification of potent drug candidates inhibiting the ERK/MAPK and associated pathways would pave a way for developing the targeted therapeutics for a subset of gastric tumors with activated ERK/MAPK signaling cascade.


Assuntos
Sistema de Sinalização das MAP Quinases/genética , Terapia de Alvo Molecular , Neoplasias Gástricas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia
15.
Proc Natl Acad Sci U S A ; 117(43): 26812-26821, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33033228

RESUMO

The expression of genes encoding powerful developmental regulators is exquisitely controlled, often at multiple levels. Here, we investigate developmental expression of three conserved genes, Caenorhabditis elegans mpk-1, lag-1, and lag-3/sel-8, which encode homologs of ERK/MAPK and core components of the Notch-dependent transcription complex, respectively. We use single-molecule FISH (smFISH) and MATLAB to visualize and quantify nuclear nascent transcripts and cytoplasmic mRNAs as a function of position along the germline developmental axis. Using differentially labeled probes, one spanning an exceptionally long first intron and the other spanning exons, we identify two classes of active transcription sites (ATS). The iATS class, for "incomplete" ATS, harbors only partial nascent transcripts; the cATS class, for "complete" ATS, harbors full-length nascent transcripts. Remarkably, the frequencies of iATS and cATS are patterned along the germline axis. For example, most mpk-1 ATS are iATS in hermaphrodite germline stem cells, but most are cATS in differentiating stem cell daughters. Thus, mpk-1 ATS class frequencies switch in a graded manner as stem cell daughters begin differentiation. Importantly, the patterns of ATS class frequency are gene-, stage-, and sex-specific, and cATS frequency strongly correlates with transcriptional output. Although the molecular mechanism underlying ATS classes is not understood, their primary difference is the extent of transcriptional progression. To generate only partial nascent transcripts in iATS, progression must be slowed, paused, or aborted midway through the gene. We propose that regulation of ATS class can be a critical mode of developmental gene regulation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Software , Fatores de Transcrição/genética
16.
Ecotoxicol Environ Saf ; 267: 115651, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37913581

RESUMO

While existing research has illuminated the environmental dangers and neurotoxic effects of MC-LR exposure, the molecular underpinnings of brain damage from environmentally-relevant MC-LR exposure remain elusive. Employing a comprehensive approach involving RNA sequencing, histopathological examination, and biochemical analyses, we discovered genes differentially expressed and enriched in the ferroptosis pathway. This finding was associated with mitochondrial structural impairment and downregulation of Gpx4 and Slc7a11 in mice brains subjected to low-dose MC-LR over 180 days. Mirroring these findings, we noted reduced cell viability and GSH/GSSH ratio, along with an increased ROS level, in HT-22, BV-2, and bEnd.3 cells following MC-LR exposure. Intriguingly, MC-LR also amplified phospho-Erk levels in both in vivo and in vitro settings, and the effects were mitigated by treatment with PD98059, an Erk inhibitor. Taken together, our findings implicate the activation of the Erk/MAPK signaling pathway in MC-LR-induced ferroptosis, shedding valuable light on the neurotoxic mechanisms of MC-LR. These insights could guide future strategies to prevent MC-induced neurodegenerative diseases.


Assuntos
Células Endoteliais , Ferroptose , Camundongos , Animais , Encéfalo , Transdução de Sinais
17.
Chem Biodivers ; 20(7): e202300050, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37314937

RESUMO

BACKGROUND: Liver cancer is an extremely common cancer with the highest mortality rate and poor prognosis. Owing to their low systemic toxicity and few side effects, natural compounds may provide better therapeutic effects for patients. (2E)-1-(2,4,6-trimethoxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (TMOCC), a chalcone derivative, exhibits cytotoxicity towards many tumor cells. However, the anticancer mechanism of TMOCC has not been elucidated in human hepatocellular carcinoma (HCC). METHODS: Cell Counting Kit-8 and colony formation assays were used to evaluate the effects of TMOCC on viability and proliferation. Mitochondrial transmembrane potential and flow cytometry assays were used to detect apoptosis. The expression levels of proteins related to apoptosis, the RAS-ERK and AKT/FOXO3a signaling pathways were assessed using western blot. Potential targets of TMOCC were detected using molecular docking analysis. RESULTS: TMOCC inhibited viability and proliferation, and induced the loss of mitochondrial transmembrane potential, apoptosis and DNA double-strand breaks in both HCC cells. The RAS-ERK and AKT/FOXO3a signaling pathways were suppressed by TMOCC. Finally, ERK1, PARP-1, and BAX were identified as potential targets of TMOCC. CONCLUSION: Taken together, our results show that TMOCC promotes apoptosis by suppressing the RAS-ERK and AKT/FOXO3a signaling pathways. TMOCC may be a potential multi-target compound that is effective against liver cancer.


Assuntos
Carcinoma Hepatocelular , Chalcona , Chalconas , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Chalconas/farmacologia , Chalconas/uso terapêutico , Chalcona/farmacologia , Simulação de Acoplamento Molecular , Apoptose , Linhagem Celular Tumoral , Proliferação de Células
18.
J Cell Mol Med ; 26(23): 5832-5845, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36377725

RESUMO

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase-mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB-Med-8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC-0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti-migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.


Assuntos
Neoplasias Cerebelares , Meduloblastoma , Proteínas rac1 de Ligação ao GTP , Humanos , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas rac1 de Ligação ao GTP/metabolismo , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico
19.
Genes Cells ; 26(2): 109-116, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33249692

RESUMO

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.


Assuntos
Apoptose/efeitos dos fármacos , Álcoois Benzílicos/farmacologia , Regulação para Baixo/genética , Fosfatase 6 de Especificidade Dupla/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Animais , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fosfatase 6 de Especificidade Dupla/metabolismo , Camundongos , Células NIH 3T3 , Oncogenes
20.
Reprod Biol Endocrinol ; 20(1): 159, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36401313

RESUMO

BACKGROUND: The widely accepted explanation of preeclampsia (PE) pathogenesis is insufficient trophoblast invasion and impaired uterine spiral artery remodeling. However, the underlying molecular mechanism remains unclear. METHODS: We performed transcriptome sequencing on placentas of normal and PE patients and identified 976 differentially expressed long noncoding RNAs (lncRNAs). TCF21 antisense RNA inducing demethylation (TARID) was one of the most significantly differentially expressed lncRNAs and was negatively correlated with the systolic and diastolic blood pressure in PE patients. Furthermore, we verified the effect of TARID on the biological behavior of trophoblasts and performed UID mRNA-seq to identify the effectors downstream of TARID. Then, co-transfection experiments were used to better illustrate the interaction between TARID and its downstream effector. RESULTS: We concluded that the downregulation of TARID expression may inhibit trophoblast infiltration and spiral artery remodeling through inhibition of cell migration, invasion, and tube formation mediated through the CXCL3/ERK/MAPK pathway. CONCLUSIONS: Overall, these findings suggested that TARID may be a therapeutic target for PE through the CXCL3/ERK/MAPK pathway.


Assuntos
Pré-Eclâmpsia , RNA Longo não Codificante , Humanos , Gravidez , Feminino , Trofoblastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Pré-Eclâmpsia/etiologia , RNA Antissenso/metabolismo , Proliferação de Células/genética , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo
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