RESUMO
G0S2 and HIG2 are two selective inhibitors of ATGL (also known as PNPLA2), the key enzyme for intracellular lipolysis. Whereas G0S2 regulates triglyceride (TG) mobilization in adipocytes and hepatocytes, HIG2 functions to enhance intracellular TG accumulation under hypoxic conditions. A homologous hydrophobic domain (HD) is shared by G0S2 and HIG2 (also known as HILPDA) for binding to ATGL. However, the determinants of their lipid droplet (LD) localization are unknown. Here, we study how G0S2 and HIG2 are targeted to LDs, and identify both ATGL-independent and -dependent mechanisms. Structural prediction and studies in cells reveal that ATGL-independent localization of G0S2 to both the endoplasmic reticulum (ER) and LDs is mediated by a hairpin structure consisting of two hydrophobic sequences. Positively charged residues in the hinge region play a crucial role in sorting G0S2, which initially localizes to ER, to LDs. Interestingly, the role of these positive charges becomes dispensable when ATGL is co-expressed. In comparison, HIG2, which lacks a similar hairpin structure, is dependent on ATGL for its full LD targeting. Thus, our studies identify specific structural features and mechanisms for mediating accumulation of these two ATGL inhibitors on LDs.
Assuntos
Gotículas Lipídicas , Lipólise , Gotículas Lipídicas/metabolismo , Lipase/genética , Lipase/metabolismo , Adipócitos/metabolismo , Metabolismo dos LipídeosRESUMO
Ischemiaâreperfusion (I/R) is a common complication in the clinical treatment of acute myocardial infarction (MI), in which cardiomyocytes play a pivotal role in the recovery of cardiac function after reperfusion injury. The expression of numerous circular ribonucleic acids (circRNAs) is disrupted in I/R-induced cardiac damage, but the potential role of circRNAs in I/R damage has not been fully elucidated. The purpose of the present study was to clarify the biological action and molecular mechanism of circRNA 002166 (also termed circCL2L13) in postmyocardial I/R. Oxygen-glucose deprivation/reoxygenation (OGD/R) in an in vivo model was performed to simulate I/R damage. real-time polymerase chain reaction analysis was also conducted to evaluate the relationships of the SOD1, SOD2, NRF2, HO1 and GPX4 indicators with oxidative stress injury. TUNEL immunofluorescence was used to evaluate the degree of cardiomyocyte apoptosis in the different treatment groups. The circBCL2L13 level was markedly upregulated in myocardial tissues from a mouse I/R model. Overexpression of circBCL2L13 markedly attenuated the expression of oxidative stress-related genes and apoptosis in OGD/R-induced cardiomyocytes. A mechanistic study revealed that circBCL2L13 functions as a ceRNA for miR-1246 and modulates paternally expressed gene 3 (PEG3). Eventually, circBCL2L13 was proven to regulate PEG3 by targeting miR-1246, thereby protecting against OGD/R-induced cardiomyocyte oxidative damage and apoptosis. In conclusion, our study confirmed that the circBCL2L13/miR-1246/PEG3 axis suppressed the progression of OGD/R injury in cardiomyocytes, which might lead to new therapeutic strategies for cardiac I/R injury.
Assuntos
Apoptose , MicroRNAs , Estresse Oxidativo , RNA Circular , Traumatismo por Reperfusão , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Traumatismo por Reperfusão/metabolismo , RNA Circular/genética , RNA Circular/metabolismoRESUMO
Non-small cell lung cancer (NSCLC) patients are characterized by distant metastasis and poor prognosis. Growing evidence has implied that circular RNAs (circRNAs) are involved in multiple tumor progression, including NSCLC. The objective of the present study was to functionally dissect the role and mechanism of circ_BLNK in NSCLC development and progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of circ_BLNK, miR-942-5p, and forkhead box protein O1 (FOXO1) in NSCLC tissues and cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay detected cell proliferation; the protein expression levels were tested by western blot assay; cell apoptosis was measured by flow cytometry, and transwell assay detected cell migration and invasion. The molecular targeting relationship was determined by dual-luciferase reporter assay. The effect of circ_BLNK overexpression on tumor growth was detected by in vivo experiments and immunohistochemistry. Circ_BLNK was dramatically decreased in NSCLC, and overexpression of circ_BLNK inhibited proliferation, migration, and invasion of NSCLC cells and promoted cell apoptosis. Circ_BLNK level was negatively correlated with miR-942-5p expression and positively correlated with FOXO1 expression. Moreover, circ_BLNK acted as a sponge for miR-942-5p, which targeted FOXO1. Rescue assays presented that miR-942-5p reversed the anticancer action of circ_BLNK in NSCLC. Besides that, miR-942-5p inhibition suppressed the oncogenic behaviors, which were attenuated by FOXO1 knockdown. Animal experiments exhibited that circ_BLNK upregulation repressed tumor growth in vivo. Our study demonstrated a novel regulatory mechanism that circ_BLNK/miR-942-5p/FOXO1 axis adjusted non-small cell lung cancer development.
RESUMO
BACKGROUND & AIMS: The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS: The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS: In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS: Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/metabolismo , Ceramidas/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Hipóxia/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of human lung cancers, which has diverse pathological features. Although many signaling pathways and therapeutic targets have been defined to play important roles in NSCLC, limiting efficacies have been achieved. METHODS: Bioinformatics methods were used to identify differential long non-coding RNA expression in NSCLC. Real-time RT-PCR experiments were used to examine the expression pattern of lncRNA PKMYT1AR, miR-485-5p. Both in vitro and in vivo functional assays were performed to investigate the functional role of PKMYT1AR/miR-485-5p/PKMYT1 axis on regulating cell proliferation, migration and tumor growth. Dual luciferase reporter assay, fluorescent in situ hybridization (FISH), immunoblot, co-immunoprecipitation experiments were used to verify the molecular mechanism. RESULT: Here, we identify a human-specific long non-coding RNA (lncRNA, ENST00000595422), termed PKMYT1AR (PKMYT1 associated lncRNA), that is induced in NSCLC by Yin Yang 1 (YY1) factor, especially in cancerous cell lines (H358, H1975, H1299, H1650, A549 and SPC-A1) compared to that in normal human bronchial epithelium cell line (BEAS-2B). We show that PKMYT1AR high expression correlates with worse clinical outcome, and knockdown of PKMYT1AR inhibits tumor cell proliferation, migration and xenograft tumor formation abilities. Bioinformatic analysis and a luciferase assay demonstrate that PKMYT1AR directly interacts with miR-485-5p to attenuate the inhibitory role on its downstream oncogenic factor PKMYT1 (the protein kinase, membrane-associated tyrosine/threonine 1) in NSCLC. Furthermore, we uncover that miR-485-5p is downregulated in both cancerous cell lines and peripheral blood serum isolated from NSCLC patients compared to reciprocal control groups. Consistently, forced expression of miR-485-5p inhibits the proliferation and migration abilities of tumor cells. Moreover, we provide evidence showing that PKMYT1AR targeting antisense oligonucleotide (ASO) dramatically inhibit tumor growth in vivo. Mechanistic study shows that PKMYT1AR/ miR-485-5p /PKMYT1 axis promotes cancer stem cells (CSCs) maintenance in NSCLC via inhibiting ß-TrCP1 mediated ubiquitin degradation of ß-catenin proteins, which in turn causes enhanced tumorigenesis. CONCLUSIONS: Our findings reveal the critical role of PKMYT1AR/miR-485-5p /PKMYT1 axis during NSCLC progression, which could be used as novel therapeutic targets in the future.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/etiologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , RNA Longo não Codificante/genética , Via de Sinalização Wnt , Regiões 3' não Traduzidas , Animais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , MicroRNAs , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso , Prognóstico , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estabilidade Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Interferência de RNARESUMO
Historically, the Cambrian explosion was a major life evolution event caused by changes of natural environmental oxygen concentration. The use of oxygen was part of the basic survival instinct of higher life, which evolved a complex regulation system in response to variant levels of oxygen concentration. Hypoxia is one of the typical environmental characteristics in plateau areas. After long-term natural selection in hypoxic conditions, numerous species living in plateau areas have evolved unique mechanisms adapted to hypoxia. Recent studies have found that there are some similarities in adaptation to hypoxia between the animals in highland and different types of human solid tumor cells. Herein, we will summarize recent findings about the hypoxia adaptation evolution in high-altitude animals and the characteristics of hypoxic solid tumors, especially the reactive oxygen species responses in hypoxic solid tumors. We believe that deciphering the underlying molecular mechanisms involved in hypoxia adaptation in highland will facilitate the identification of new genes or biomarkers critical for research on hypoxic solid tumors in the future.
Assuntos
Doença da Altitude , Altitude , Aclimatação , Animais , Humanos , Hipóxia , Oxigênio , Seleção GenéticaRESUMO
BACKGROUND: Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a key mechanism in pulmonary arterial hypertension (PAH). Serotonin (5-hydroxytryptamine, 5-HT) can induce abnormal proliferation of PASMCs. The role of miR-361-3p in serotonin-induced abnormal PASMCs proliferation remains unclear. METHODS: The miR-361-3p level was analyzed in plasma from PAH patients and normal controls and in human PASMCs (hPASMCs) using RT-PCR. The hPASMCs were transfected with an miR-361-3p mimic and then treated with serotonin. Untransfected hPASMCs were used as the control. Cell proliferation was evaluated using an MTS assay and 5-ethynyl-2'-deoxyuridine (EdU) staining. The cell cycle stages were evaluated using flow cytometry. The association between miR-361-3p and serotonin transporter (SERT) was determined using a luciferase reporter assay and anti-AGO2 RNA immunoprecipitation assay. The protein expression was evaluated via western blotting. RESULTS: The miR-361-3p level was lower in plasma from PAH patients than in plasma from the any of the normal control subjects. The mean pulmonary arterial pressure, pulmonary vascular resistance and pulmonary vascular resistance index were higher in PAH patients whose miR-361-3p level was lower than the median value for patients than in those whose miR-361-3p level was higher than the median. Serotonin treatment reduced miR-361-3p expression in the hPASMCs. MiR-361-3p overexpression suppressed cell proliferation, promoted apoptosis, induced G1 arrest, and decreased the phosphorylation level of ERK1/2 in serotonin-treated hPASMCs. SERT was identified as an miR-361-3p target. Its overexpression alleviated the effect of miR-361-3p overexpression on serotonin-induced hPASMC proliferation and upregulation of phosphorylated ERK1/2. CONCLUSIONS: The miR-361-3p level is lower in the plasma of PAH patients. Upregulation of miR-361-3p suppresses serotonin-induced proliferation of hPASMCs by targeting SERT. Our results suggest that miR-361-3p is a potential therapeutic target in PAH.
Assuntos
Proliferação de Células/efeitos dos fármacos , MicroRNAs/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Regulação para Cima/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Fase G1/genética , Células HEK293 , Humanos , Pulmão/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Serotonina/farmacologia , Transdução de Sinais/genéticaRESUMO
Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.
Assuntos
Caseína Quinase I/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Western Blotting , Caseína Quinase I/genética , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Hedgehog/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Fosforilação , Interferência de RNA , Receptor Smoothened/genética , Asas de Animais/metabolismoRESUMO
At cocktail parties, our brains often simultaneously receive visual and auditory information. Although the cocktail party problem has been widely investigated under auditory-only settings, the effects of audiovisual inputs have not. This study explored the effects of audiovisual inputs in a simulated cocktail party. In our fMRI experiment, each congruent audiovisual stimulus was a synthesis of 2 facial movie clips, each of which could be classified into 1 of 2 emotion categories (crying and laughing). Visual-only (faces) and auditory-only stimuli (voices) were created by extracting the visual and auditory contents from the synthesized audiovisual stimuli. Subjects were instructed to selectively attend to 1 of the 2 objects contained in each stimulus and to judge its emotion category in the visual-only, auditory-only, and audiovisual conditions. The neural representations of the emotion features were assessed by calculating decoding accuracy and brain pattern-related reproducibility index based on the fMRI data. We compared the audiovisual condition with the visual-only and auditory-only conditions and found that audiovisual inputs enhanced the neural representations of emotion features of the attended objects instead of the unattended objects. This enhancement might partially explain the benefits of audiovisual inputs for the brain to solve the cocktail party problem.
Assuntos
Percepção Auditiva/fisiologia , Encéfalo/fisiologia , Resolução de Problemas/fisiologia , Percepção Visual/fisiologia , Adulto , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Choro/psicologia , Emoções , Humanos , Processamento de Imagem Assistida por Computador , Riso/psicologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiologia , Reprodutibilidade dos Testes , Meio Social , Adulto JovemRESUMO
G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. Here we provide evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Drosophila/enzimologia , Proteínas de Drosophila/genética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Proteínas Hedgehog/genética , Fosforilação , Receptores Acoplados a Proteínas G/genética , Receptor SmoothenedRESUMO
In this study, we evaluated the prognostic potential and functional regulation of human nature antisense, brain-derived neurotrophic factor antisense, in non-small cell lung cancer. Non-small cell lung cancer carcinoma and adjacent non-carcinoma lung tissues were extracted from 151 patients. Their endogenous brain-derived neurotrophic factor antisense expression levels were compared by quantitative reverse transcription polymerase chain reaction. Clinical relevance between endogenous brain-derived neurotrophic factor antisense expression level and patients' clinicopathological variances or overall survival was analyzed. The potential of brain-derived neurotrophic factor antisense being an independent prognostic factor in non-small cell lung cancer was also evaluated. In in vitro non-small cell lung cancer cell lines, brain-derived neurotrophic factor antisense was upregulated through forced overexpression. The effects of brain-derived neurotrophic factor antisense upregulation on non-small cell lung cancer in vitro survival, proliferation, and migration were evaluated by viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and transwell assays. Brain-derived neurotrophic factor antisense is lowly expressed in non-small cell lung cancer carcinoma tissues and further downregulated in late-stage carcinomas. Brain-derived neurotrophic factor antisense downregulation was closely associated with non-small cell lung cancer patients' advanced tumor, lymph node, metastasis stage, and positive status of lymph node metastasis, and confirmed to be an independent prognostic factor for patients' poor overall survival. In non-small cell lung cancer A549 and H226 cell lines, forced overexpression of brain-derived neurotrophic factor antisense did not alter cancer cell viability but had significantly tumor suppressive effect in inhibiting in vitro non-small cell lung cancer proliferation and migration. Endogenous brain-derived neurotrophic factor antisense in non-small cell lung cancer carcinoma could be a potential biomarker for predicting patients' prognosis. Overexpressing brain-derived neurotrophic factor antisense may also have a therapeutic potential in inhibiting non-small cell lung cancer tumor growth.
Assuntos
Biomarcadores Tumorais/genética , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Prognóstico , RNA Longo não Codificante/genética , Células A549 , Idoso , Biomarcadores Tumorais/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , RNA Antissenso/biossíntese , RNA Antissenso/genética , RNA Longo não Codificante/biossínteseRESUMO
Hedgehog (Hh) signaling governs many developmental processes by regulating the balance between the repressor (Ci(R)/Gli(R)) and activator (Ci(A)/Gli(A)) forms of Cubitus interruptus (Ci)/glioma-associated oncogene homolog (Gli) transcription factors. Although much is known about how Ci(R)/Gli(R) is controlled, the regulation of Ci(A)/Gli(A) remains poorly understood. Here we demonstrate that Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 and Suppressor of fused (Sufu) by protecting Ci(A) from premature degradation. We show that Hh stimulates Ci phosphorylation by CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by the Hh-induced MATH and BTB domain containing protein (HIB), a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases. In Hh-receiving cells, reduction of CK1 activity accelerated HIB-mediated degradation of Ci(A), leading to premature loss of pathway activity. We also provide evidence that Gli(A) is regulated by CK1 in a similar fashion and that CK1 acts downstream of Sufu to promote Sonic hedgehog signaling. Taken together, our study not only reveals an unanticipated and conserved mechanism by which phosphorylation of Ci/Gli positively regulates Hh signaling but also provides the first evidence, to our knowledge, that substrate recognition by the Cullin 3 family of E3 ubiquitin ligases is negatively regulated by a kinase.
Assuntos
Caseína Quinase I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caseína Quinase I/genética , Linhagem Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Hedgehog/genética , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Oncogênicas/genética , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Proteólise , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
OBJECTIVES: To search for more effective radiation protectors with minimal toxicity, a water-soluble nitroxides Acetamido-Tempol (AA-Tempol) was evaluated for potential radioprotective properties in HUVEC cells (Human Umbilical Vein Endothelial cell line). METHODS: To study the anti-radiation effect of AA-Tempol in cell culture, the viability of irradiated HUVEC cells using a clonogenic survival assay was examined. The anti-apoptosis effects of AA-Tempol using Annexin V/propidium iodide staining in a flow cytometry assay was also evaluated. To elucidate the molecular mechanism of the anti-apoptosis effect of AA-Tempol against X-radiation induced HUVEC cell apoptosis, the expression of Bax, Bcl-2 and p53 and caspase-3 were examined. The changes in the level of malondialdehyde (MDA) and glutathione (GSH) in HUVEC cells after X-radiation were also investigated. RESULTS: Pretreatment of the HUVEC cells colony with AA-Tempol 1 h before X-radiation significantly increased the colony survival (p < 0.05) compared with the cells without pretreatment. This demonstrates that AA-Tempol provides an effective radiation protection in the irradiated HUVEC cells, thus reducing apoptosis from 20.1 ± 1.3% in 8 Gy X-radiated cells to 12.2 ± 0.9% (1.0 mmol/L-1 AA-Tempol) in AA-Tempo pretreated HUVEC cells. This implies that 1.0 mM AA-Tempol treatment significantly block the increase of caspase-3 activity in radiated HUVEC cells (P < 0.01), causing down-regulation in expressions of Bax and P53 and up-regulation in the expression of Bcl-2. Pretreatment with AA-Tempol also decreased the MDA activities (P < 0.01) and increase the GSH level (P < 0.05) in HUVEC cells compared to the 8Gy X-radiated cells without pretreatment. CONCLUSIONS: These observations indicate that AA-Tempol is a potential therapeutic agent against the radiation damage.
Assuntos
Óxidos N-Cíclicos/farmacologia , Protetores contra Radiação/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Metastasis is a crucial hallmark of cancer progression, which involves numerous factors including the degradation of the extracellular matrix (ECM), the epithelial-to-mesenchymal transition (EMT), tumor angiogenesis, the development of an inflammatory tumor microenvironment, and defects in programmed cell death. Programmed cell death, such as apoptosis, autophagy, and necroptosis, plays crucial roles in metastatic processes. Malignant tumor cells must overcome these various forms of cell death to metastasize. This review summarizes the recent advances in the understanding of the mechanisms by which key regulators of apoptosis, autophagy, and necroptosis participate in cancer metastasis and discusses the crosstalk between apoptosis, autophagy, and necroptosis involved in the regulation of cancer metastasis.
Assuntos
Apoptose , Autofagia , Necrose , Neoplasias/metabolismo , Neoplasias/patologia , Animais , HumanosRESUMO
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual ß-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila , Proteínas Hedgehog/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitinação/fisiologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Membrana Celular/genética , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Expressão Gênica , Inativação Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Receptor Smoothened , Distribuição Tecidual , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/fisiologiaRESUMO
BACKGROUND: To uncover the genes involved in the development of osteosarcoma (OS), we performed a meta-analysis of OS microarray data to identify differentially expressed genes (DEGs) and biological functions associated with gene expression changes between OS and normal control (NC) tissues. METHODS: We used publicly available GEO datasets of OS to perform a meta-analysis. We performed Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Protein-Protein interaction (PPI) networks analysis. RESULTS: Eight GEO datasets, including 240 samples of OS and 35 samples of controls, were available for the meta-analysis. We identified 979 DEGs across the studies between OS and NC tissues (472 up-regulated and 507 down-regulated). We found GO terms for molecular functions significantly enriched in protein binding (GO: 0005515, P = 3.83E-60) and calcium ion binding (GO: 0005509, P = 3.79E-13), while for biological processes, the enriched GO terms were cell adhesion (GO:0007155, P = 2.26E-19) and negative regulation of apoptotic process (GO: 0043066, P = 3.24E-15), and for cellular component, the enriched GO terms were cytoplasm (GO: 0005737, P = 9.18E-63) and extracellular region (GO: 0005576, P = 2.28E-47). The most significant pathway in our KEGG analysis was Focal adhesion (P = 5.70E-15). Furthermore, ECM-receptor interaction (P = 1.27E-13) and Cell cycle (P = 4.53E-11) are found to be highly enriched. PPI network analysis indicated that the significant hub proteins containing PTBP2 (Degree = 33), RGS4 (Degree = 15) and FXYD6 (Degree = 13). CONCLUSIONS: Our meta-analysis detected DEGs and biological functions associated with gene expression changes between OS and NC tissues, guiding further identification and treatment for OS.
Assuntos
Regulação da Expressão Gênica , Osteossarcoma/genética , Mapas de Interação de Proteínas/genética , Proteínas de Ligação ao Cálcio/genética , Ciclo Celular/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/genéticaRESUMO
Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.
Assuntos
Caseína Quinase Ialfa/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cílios/metabolismo , Drosophila/genética , Drosophila/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação , Transdução de Sinais , Receptor Smoothened , TransfecçãoRESUMO
The objective of this study was to provide a precise evaluation of whether expression levels of excision repair cross-complementation group 1 (ERCC1) are associated with objective response, overall survival (OS), and median survival in patients with advanced bladder cancer treated with platinum-based chemotherapy. Systematic computerized searches of the electronic databases PubMed, EMBASE, Ovid, ASCO, and CNKI were performed and a meta-analysis was carried out to evaluate the correlation between ERCC1 expression levels and objective response rate, OS, or progression-free survival in patients with advanced bladder cancer receiving platinum-based chemotherapy. References within the articles identified were also searched manually. STATA package version 11.0 was used for the comprehensive quantitative analyses. A total of six studies involving 356 patients, of which ERCC1 expression was high/positive in 138 (38.8%) and low/negative in 218 (61.2%), were included in the meta-analysis. The median age of the patients was 63.7 years. The objective response rate favored patients with ERCC1 low/negative expression after platinum-based chemotherapy, but showed no significant difference [odds ratio 0.86, 95% confidence interval (CI) 0.36-2.06, P=0.734]. The median OS time and the median progression-free survival time were significantly prolonged when ERCC1 low/negative expression was compared with ERCC1 high/positive expression (hazard ratio 0.69, 95% CI 0.54-0.89, P=0.004, and hazard ratio 0.76, 95% CI 0.66-0.89, P=0.000, respectively). In conclusion, low/negative expression of ERCC1 was associated with higher objective response, median progression-free survival, and median OS in patients with advanced bladder cancer treated with platinum-based chemotherapy. ERCC1 may be a suitable marker of prognosis and sensitivity to platinum-based chemotherapy in patients with advanced bladder cancer. Larger studies and further clinical trials are warranted to confirm these findings.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Platina/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Endonucleases/genética , Humanos , Neoplasias da Bexiga Urinária/metabolismoRESUMO
OBJECTIVE: To investigate the effects of electromagnetic pulses (EMP) on the apoptosis and transforming growth factor beta 3 (TGF-ß3) expression of mouse testis tissue. METHODS: Thirty-two male BALB/c mice were randomly and equally divided into one control group and three EMP treated groups, which were whole-body exposed to EMP at 200 kV/m with 100, 200, and 400 pulses, respectively. The control group received no treatment. The pathological changes and cell apoptosis in testis tissue were analyzed by TUNEL assay. The mRNA expression of TGF-ß3 in testis tissue was determined by RT-PCR, and the protein expression of TGF-ß3 was determined by immunohistochemistry and Western blot. RESULTS: No obvious pathological changes were found in testis tissue after EMP exposure at 200 kV/m with 100 and 200 pulses. However, after EMP exposure with 400 pulses, degeneration and shedding of testis tissue, accompanied by significant increase in apoptosis rate (P < 0.05), was observed. The RT-PCR, immunohistochemistry, and Western blot showed that the expression of TGF-ß3 mRNA and protein increased significantly after EMP exposure with 400 pulses as compared with that of the control group (P < 0.05). CONCLUSION: EMP exposure at 200 kV/m with 400 pulses increases the incidence of apoptosis and expression of TGF-ß3 in mouse testis tissue, which is potentially one of the mechanisms by which EMP increases blood-testis barrier permeability in mice.
Assuntos
Apoptose , Campos Eletromagnéticos/efeitos adversos , Testículo/patologia , Fator de Crescimento Transformador beta3/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testículo/metabolismoRESUMO
This paper mainly investigated the effect of the Mn/Ag ratio on the microstructure and room temperature and high-temperature (350 °C) tensile mechanical properties of the as-cast and heat-treated Al-6Cu-xMn-yAg (x + y = 0.8, wt.%) alloys. The as-cast alloy has α-Al, Al2Cu, and a small amount of Al7Cu2 (Fe, Mn) and Al20Cu2 (Mn, Fe)3 phases. After T6 heat treatment, a massive dispersive and fine θ'-Al2Cu phase (100~400 nm) is precipitated from the matrix. The Mn/Ag ratio influences the quantity and size of the precipitates; when the Mn/Ag ratio is 1:1, the θ'-Al2Cu precipitation quantity reaches the highest and smallest. Compared with the as-cast alloy, the tensile strength of the heat-treated alloy at room temperature and high temperature is greatly improved. The strengthening effect of the alloy is mainly attributed to the nanoparticles precipitated from the matrix. The Mn/Ag ratio also affects the high-temperature tensile mechanical properties of the alloy. The high-temperature tensile strength of the alloy with a 1:1 Mn/Ag ratio is the highest, reaching 135.89 MPa, 42.95% higher than that of the as-cast alloy. The analysis shows that a synergistic effect between Mn and Ag elements can promote the precipitation and refinement of the θ'-Al2Cu phase, and there is an optimal ratio (1:1) that obtains the lowest interfacial energy for co-segregation of Mn and Ag at the θ'/Al interface that makes θ'-Al2Cu have the best resistance to coarsening.