RESUMEN
Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKß-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKß/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.
Asunto(s)
Arsenitos , Proteína p53 Supresora de Tumor , Apoptosis , Arsenitos/metabolismo , Arsenitos/farmacología , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Exorribonucleasas/metabolismoRESUMEN
Sleep deprivation (SD) weakens the immune system and leads to increased susceptibility to infectious or inflammatory diseases. However, it is still unclear how SD affects humoral immunity. In the present study, sleep disturbance was conducted using an sleep deprivation instrument, and the bacterial endotoxin lipopolysaccharide (LPS) was used to activate the immune response. It was found that SD-pretreatment reduced LPS-induced IgG2b+ B cells and IgG2b isotype antibody production in lymphocytes of spleen. And, SD-pretreatment decreased the proportion of CD4+T cells, production of CD4+T cells derived TGF-ß1 and its contribution in helping IgG2b production. Additionally, BMAL1 and CLOCK were selectively up-regulated in lymphocytes after SD. Importantly, BMAL1 and CLOCK deficiency contributed to TGF-ß1 expression and production of IgG2b+ B cells. Thus, our results provide a novel insight to explain the involvement of BMAL1 and CLOCK under SD stress condition, and their roles in inhibiting TGF-ß1 expression and contributing to reduction of LPS induced IgG2b production.
Asunto(s)
Factores de Transcripción ARNTL , Formación de Anticuerpos , Proteínas CLOCK , Inmunoglobulina G , Privación de Sueño , Privación de Sueño/genética , Privación de Sueño/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ratas Sprague-Dawley , Ratones Endogámicos C57BL , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/inmunología , Proteínas CLOCK/genética , Proteínas CLOCK/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/genética , Estrés Fisiológico/inmunología , Animales , Ratones , Ratas , Células CultivadasRESUMEN
Chronic stress enhances the risk for psychiatric disorders and induces depression and cognitive impairment. Gamma oscillations are essential for neurocircuit function, emotion, and cognition. However, the influence of gamma entrainment by sensory stimuli on specific aspects of chronic stress-induced responses remains unclear. Mice were subjected to corticosterone (CORT) administration and chronic restraint stress (CRS) for weeks, followed by rhythmic gamma frequency light flickering exposure. Local field potentials (LFPs) were recorded from the V1, CA1, and PFC regions to verify the light flicker on gamma oscillations. Behavioral tests were used to examine stress-related and memory-related behaviors. Golgi staining was performed to observe changes in spine morphology. Synaptosomes were isolated to determine the expression of synapse-related proteins through immunoblotting. RNA sequencing (RNA-seq) was applied to explore specific changes in the transcriptome. Immunofluorescence staining, real-time quantitative polymerase chain reaction (qPCR), and ELISA were used to evaluate microglial activation and cytokine levels. In this study, we demonstrated that rhythmic 40 Hz LF attenuated stress-related behavior and cognitive impairments by ameliorating the microstructural alterations in spine morphology and increasing the expression of GluN2A and GluA1 in chronically stressed mice. Transcriptome analysis revealed that significantly downregulated genes in LF-exposed CRS mice were enriched in neuroimmune-related signaling pathways. Rhythmic 40 Hz LF exposure significantly decreased the number of Iba1-positive microglia in the PFC and hippocampus, and the expression levels of the M1 markers of microglia iNOS and CD68 were reduced significantly in CRS mice. In addition, 40 Hz LF exposure suppressed the secretion of cytokines IL-12, which could regulate the production of IFN-γ and IL-10 in stressed mice. Our results demonstrate that exposure to rhythmic 40 Hz LF induces the neuroimmune response and downregulation of neuroinflammation with attenuated stress-related behaviors and cognitive function in CRS-induced mice. Our findings highlight the importance of sensory-evoked gamma entrainment as a potential therapeutic strategy for stress-related disorders treatment. Abbreviations: CORT, Chronic corticosterone treatment; CRS, Chronic restraint stress; IACUC, Institutional Animal Care and Use Committee; LF, light flickers; FST, Forced swim test; NSFT, Novelty-suppressed feeding test; SPT, Sucrose preference test; NSFT, Novelty-suppressed feeding; qPCR, Quantitative real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline plus 0.1% Tween 20; PVDF, polyvinylidene fluoride; GFAP, Glial fibrillary acidic protein; DAPI, 4',6-Diamid- ino-2-phenylindole; Iba1, Ionized calcium-binding adaptor molecule 1; iNOS, Inducible nitric oxide synthase; IL-10, Interleukin-10; IL6, Interleukin 6; IL-1ß, Interleukin 1ß; IL-12, Interleukin 12; TNF-α, Tumor necrosis factor alpha; IFN-γ, Interferon-gamma; TLR6 and 9, Toll-like Receptor 6 and 9.
RESUMEN
Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas de Unión al ARN/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/metabolismo , Conversión Génica , Eliminación de Gen , Duplicación de Gen , Humanos , Modelos Biológicos , Unión Proteica , Recombinasa Rad51/metabolismoRESUMEN
Particulate matter (PM) 2.5 has long been regarded as a major risk factor of the respiratory system, which constitutes a threat to human health. Although the positive relationship between PM2.5 exposure and the development of respiratory diseases has been well established, limited studies investigate the intrinsic self-protection mechanisms against PM2.5-induced respiratory injuries. Excessive pulmonary inflammation served as a key pathogenic mechanism in PM2.5-induced airway dysfunction, and we have previously shown that PM2.5 induced the production of vascular endothelial growth factor A (VEGFA) in the bronchial epithelial cells, which subsequently led to pulmonary inflammatory responses. In the current study, we found that PM2.5 also concurrently induced the expression of the stress-responsive protein heme oxygenase-1 (HO-1) along with VEGFA in the bronchial epithelial cells both in vivo and in vitro. Importantly, knocking down of HO-1 expression significantly increased the synthesis and secretion of VEGFA; while overexpression of HO-1 showed the opposite effects, indicating that HO-1 induction can antagonize VEGFA production in the bronchial epithelial cells upon PM2.5 exposure. Mechanistically, HO-1 inhibited PM2.5-evoked VEGFA induction through modulating hypoxia-inducible factor 1 alpha (HIF-1α), which was the upstream transcriptional factor of VEGFA. More specifically, HO-1 could not only inhibit HIF-1α expression, but also suppress its transactivity. Taken together, our results suggested that HO-1 was an intrinsic protective factor against PM2.5-induced pulmonary VEGFA production with a mechanism relating to HIF-1α, thus providing a potential treatment strategy against PM2.5 triggered airway injuries.
Asunto(s)
Hemo-Oxigenasa 1 , Factor A de Crecimiento Endotelial Vascular , Humanos , Hemo-Oxigenasa 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pulmón/metabolismo , Células Epiteliales/metabolismo , Material Particulado/toxicidad , Subunidad alfa del Factor 1 Inducible por HipoxiaRESUMEN
In our previous report, we demonstrated that one of the catalytic subunits of the IκB kinase (IKK) complex, IKKα (encoded by CHUK), performs an NF-κB-independent cytoprotective role in human hepatoma cells under the treatment of the anti-tumor therapeutic reagent arsenite. IKKα triggers its own degradation, as a feedback loop, by activating p53-dependent autophagy, and therefore contributes substantially to hepatoma cell apoptosis induced by arsenite. Interestingly, IKKα is unable to interact with p53 directly but plays a critical role in mediating p53 phosphorylation (at Ser15) by promoting CHK1 activation and CHK1-p53 complex formation. In the current study, we found that p53 acetylation (at Lys373 and/or Lys382) was also critical for the induction of autophagy and the autophagic degradation of IKKα during the arsenite response. Furthermore, IKKα was involved in p53 acetylation through interaction with the acetyltransferases for p53, p300 (also known as EP300) and CBP (also known as CREBBP) (collectively p300/CBP), inducing CHK1-dependent p300/CBP activation and promoting p300-p53 or CBP-p53 complex formation. Therefore, taken together with the previous report, we conclude that both IKKα- and CHK1-dependent p53 phosphorylation and acetylation contribute to mediating selective autophagy feedback degradation of IKKα during the arsenite-induced proapoptotic responses.
Asunto(s)
Quinasa I-kappa B , Proteína p53 Supresora de Tumor , Acetilación , Autofagia , Retroalimentación , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Biogenic manganese oxides (BioMnOx) have been found all over the world, and most of them were formed by Mn(II)-oxidizing bacteria (MnOB). In this study, a MnOB designated as FF-1 was isolated from marine surface sediments in the Bohai Sea, China. This strain was identified as Bacillus sp. and can tolerate more than 5% salinity. It can grow in the presence of 0-7 mM Mn(II) and pH range from 5.0 to 7.0. When the initial Mn(II) was 5 mM, the percentage of Mn(II) oxidation reached the highest value of 16% after 10 days of incubation. The initial pH (5.0 to 7.0) affected the percentage of Mn(II) oxidation, but the ability of the strain FF-1 to self-regulate pH resulted in the final pH being almost 7.6. The removal of Mn(II) by the strain FF-1 involves extracellular and intracellular adsorption as well as Mn(II) oxidation. Intracellular Mn adsorption contributed a small part to the total Mn removal, and extracellular adsorption was dominant in the initial stage of Mn removal. The solid products after Mn removal were a mixture of MnOx and MnCO3. The layered MnOx formed in the extracellular space could be easily collected and used for adsorption and oxidation of pollutants.
Asunto(s)
Bacillus , Contaminantes Ambientales , Bacillus/genética , Bacterias , Manganeso , Naftalenos , Oxidación-Reducción , ÓxidosRESUMEN
Ornithine transcarbamylases (OTC), a key enzyme in urea cycle, is an important marker for some liver injury or diseases. However, whether OTC could be a sensitive indicator for liver dysfunction under sleep disturbance condition remains unknown. The present study aimed to explore the circadian oscillation expression of OTC and its significance in disturbed sleep condition. Sleep disturbance was conducted by a sleep deprivation (SD) instrument. Our results found that SD for 72h induced abnormal increasing of OTC levels in serum and liver of rats. And, serum OTC concentration and liver OTC expression could return to normal levels after recovery sleep following SD. Moreover, hepatic OTC expression showed circadian oscillation in day and night, characterized with occurrence of a peak between ZT 22 and ZT 2, and a nadir between ZT 14 and ZT 18. Further analysis suggested the existence of ROR response element (RORE) for potential RORÉ binding sites in OTC promoter region, and elevated RORÉ expression in rat livers under sleep disturbance condition. Additionally, oscillation expression of OTC induced by serum shock in HepG2 cells was characterized with a peak occurred between ZT 12 and ZT 16, and RORÉ knockdown at ZT 16 significantly lowered OTC expression. The results together indicate that OTC is closely correlated with circadian clock, and could be a sensitive indicator for sleep disturbance stress.
Asunto(s)
Ritmo Circadiano , Ornitina Carbamoiltransferasa/metabolismo , Trastornos del Sueño-Vigilia/enzimología , Trastornos del Sueño-Vigilia/fisiopatología , Animales , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Células Hep G2 , Homeostasis , Humanos , Hígado/enzimología , Masculino , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Ornitina Carbamoiltransferasa/genética , Ratas Sprague-Dawley , Sueño/genética , Trastornos del Sueño-Vigilia/genéticaRESUMEN
Exposure to ultraviolet B (UVB) has been demonstrated to induce DNA damage as well as angiogenesis-related photo-damages, which are implicated in a variety of medical problems, including sunburn, photo-aging and skin cancers. However, the molecular mechanism related to UVB-induced photo-injuries remained fully elucidated. Here we revealed that one of the catalytic subunits of the IKK complex, IKKα, played a critical role in mediating UVB-induced apoptotic responses in two kinds of UVB sensitive cells, human keratinocyte (HaCat) and mouse embryonic fibroblasts (MEFs). This function of IKKα was unrelated to NF-κB activity, but was delivered by inducing phosphorylation and acetylation of p53 and upregulating the expression of the pro-apoptotic p53 target gene, PERP. Although IKKα kinase activity was required for mediating post-translational modifications and transactivation of 53 and PERP induction, IKKα did not show direct binding ability toward p53. Instead, IKKα could interact with CHK1, the protein kinase leading to p53 phosphorylation, and trigger CHK1 activation and CHK1/p53 complex formation. At the same time, IKKα could also interact with p300 and CBP, the acetyltransferases responsible for p53 acetylation, and trigger p300/CBP activation and p300/p53 or CBP/p53 complex formation under UVB exposure. Taken together, we have identified a novel NF-κB-independent role of IKKα in mediating UVB-induced apoptosis by regulating p53 pathway activation. Targeting IKKα/p53/PERP pathway might be helpful to prevent skin photo-damages induced by sunlight.
Asunto(s)
Proteína p53 Supresora de Tumor , Rayos Ultravioleta , Animales , Apoptosis , Fibroblastos/metabolismo , Genes Supresores de Tumor , Humanos , Quinasa I-kappa B , Queratinocitos , Proteínas de la Membrana , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta/efectos adversosRESUMEN
S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.
Asunto(s)
Emparejamiento Base , Proteínas de Unión al ADN/química , ADN/química , Proteínas de Escherichia coli/química , Recombinasa Rad51/química , Rec A Recombinasas/química , Proteínas de Saccharomyces cerevisiae/química , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Conformación de Ácido Nucleico , Polimerizacion , Unión Proteica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés MecánicoRESUMEN
Acute and chronic exposure to particulate matter (PM) 2.5 is associated with adverse health effect upon the cardiovascular (CV) system. However, the molecular mechanism by which PM2.5 evokes CV injuries has not been fully clarified. In our recent report, we demonstrate that exposure to PM2.5 leads to elevation of circulating angiotensin II (ANGII) levels and local expressions of angiotensinogen (AGT, the precursor of ANGII), angiotensin-converting enzyme (ACE) and ANGII type 1 receptor (AT1R) in the vascular endothelial cells, which subsequently instigates the oxidative stress and proinflammatory response in the vascular endothelium. In the present study, we disclosed that PM2.5 exposure induced the activation of the transcriptional factor AP-1 and its components, c-Jun and ATF2, in the human vascular endothelial cells. Although the DNA-binding sites for AP-1 were identified within the promoter regions of AGT, ACE and AT1R genes, RT-PCR and immunoblot assays indicated that AP-1 transactivation was only involved in AT1R upregulation, but did not affect the induction of AGT and ACE expression under the same conditions. Furthermore, ERKs and p38K functioned as the upstream protein kinases involving in AP-1 transactivation and AT1R upregulation under PM2.5 stimulation. In addition, the oxidative stress and proinflammatory responses in the PM2.5-treated vascular endothelial cells were significantly reduced when MAPKs and AP-1 activation were inhibited. Therefore, we conclude that PM2.5 exposure induces MAPK/AP-1 cascade activation, which contributes to AT1R upregulation and vascular endothelial dysfunction. Identifying novel therapeutic targets to alleviate AP-1 transactivation and restore AT1R expression may be helpful for the management of PM2.5-induced CV burden.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Material Particulado/toxicidad , Receptor de Angiotensina Tipo 1/genética , Factor de Transcripción AP-1/genética , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Adhesión Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema de Señalización de MAP Quinasas , Estrés Oxidativo/efectos de los fármacos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Sulfhydryl-containing proteins play critical roles in various physiological and biological processes, and the activities of those proteins have been reported to be susceptible to thiol oxidation. Therefore, the development of protein thiol target fluorescent probe is highly desirable. In the present work, a biotinylated coumarin fluorescence "off-on" probe SQ for selectively detecting protein thiols in biotin receptor-positive cancer cells was designed with a 2,4-dinitrobenzenesulfony as the thiol receptor. The probe exhibited dramatic fluorescence responses toward sulfhydryl-containing proteins (ovalbumin (OVA), bovine serum albumin (BSA)): up to 170-fold fluorescence enhancement with 70 nm blue-shift was observed with the addition of OVA. However, low molecular weight thiols (Cys, glutathione (GSH), Hcy) caused negligible fluorescence changes of SQ. In addition, biotin receptor-positive Hela cells displayed strong red and green fluorescence after incubation of SQ for 1 h; neither red nor green fluorescence signal could be visualized in biotin-negative normal lung Wi38 cells. These results imply that the probe has potential application in fluorescent imaging protein thiols on the surface of Hela cells.
Asunto(s)
Colorantes Fluorescentes/química , Neoplasias/patología , Compuestos de Sulfhidrilo/análisis , Línea Celular Tumoral , HumanosRESUMEN
Chemotherapeutic resistance in breast cancer, whether acquired or intrinsic, remains a major clinical obstacle. Thus, increasing tumor cell sensitivity to chemotherapeutic agents will be helpful in improving the clinical management of breast cancer. In the present study, we found an induction of HO-1 expression in doxorubicin (DOX)-treated MDA-MB-231 human breast adenocarcinoma cells, which showed insensitivity to DOX treatment. Knockdown HO-1 expression dramatically upregulated the incidence of MDA-MB-231 cell death under DOX treatment, indicating that HO-1 functions as a critical contributor to drug resistance in MDA-MB-231 cells. We further observed that DOX exposure induced a cytoprotective autophagic flux in MDA-MB-231 cells, which was dependent on HO-1 induction. Moreover, upregulation of HO-1 expression required the activation of both signal transducer and activator of transcription (STAT)3 and its upstream regulator, protein kinase Src. Abrogating Src/STAT3 pathway activation attenuated HO-1 and autophagy induction, thus increasing the chemosensitivity of MDA-MB-231 cells. Therefore, we conclude that Src/STAT3-dependent HO-1 induction protects MDA-MB-231 breast cancer cells from DOX-induced death through promoting autophagy. In the following study, we further demonstrated the contribution of Src/STAT3/HO-1/autophagy pathway activation to DOX resistance in another breast cancer cell line, MDA-MB-468, which bears a similar phenotype to MDA-MB-231 cells. Therefore, activation of Src/STAT3/HO-1/autophagy signaling pathway might play a general role in protecting certain subtypes of breast cancer cells from DOX-induced cytotoxicity. Targeting this signaling event may provide a potential approach for overcoming DOX resistance in breast cancer therapeutics.
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Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/fisiología , Hemo-Oxigenasa 1/metabolismo , Factor de Transcripción STAT3/metabolismo , Familia-src Quinasas/metabolismo , Antineoplásicos/farmacología , Autofagia/fisiología , Western Blotting , Línea Celular Tumoral , Doxorrubicina/farmacología , Femenino , Humanos , Transducción de Señal/fisiología , TransfecciónRESUMEN
Exposure to solar ultraviolet B (UVB) radiation is known to induce several pathological reactions in the skin. In these processes, upregulation of VEGF expression has been demonstrated to be important in angiogenesis-associated photodamage and even skin cancers. However, the signaling events that are responsible for VEGF induction under UVB exposure have not been fully defined. Here, we demonstrate that the regulatory subunit of the phosphoinositide 3-kinase (PI3K), p85α, plays a role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs) and mouse epithermal cells, the effect of which is unrelated to the PI3K activity. The transcriptional factor NFAT3 functions as a downstream target of p85α to mediate the induction of VEGF expression in the UVB response. Although lacking NFAT3-binding ability, p85α is required for the recruitment of NFAT3 to the NFAT-response element within the vegf promoter. Furthermore, by identifying the adjacent NFAT- and AP-1-binding sites within the vegf promoter, we also found an induced interaction between NFAT3 and one of the AP-1 components, c-Fos, after UVB irradiation. Without the aid of c-Fos, NFAT3 lost its vegf-promoter-binding ability. Taken together, our results reveal a novel PI3K-independent role for p85α in controlling VEGF induction during the cellular UVB response by regulating NFAT3 activity. Targeting p85α might be helpful for preventing UVB-induced angiogenesis and the associated photodamage.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Fibroblastos/metabolismo , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Enfermedades de la Piel/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/efectos de la radiación , Técnicas de Inactivación de Genes , Ratones , Terapia Molecular Dirigida , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-fos/genética , ARN Interferente Pequeño/genética , Elementos de Respuesta/genética , Enfermedades de la Piel/etiología , Enfermedades de la Piel/prevención & control , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Rayos Ultravioleta/efectos adversos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Trastuzumab is currently used for patients with Her2(+) advanced gastric cancer. However, the response rate to trastuzumab among the patients is low. The molecular mechanisms underlying trastuzumab resistance in gastric cancer are unknown. Our in vitro data show that activation of ß2-adrenergic receptor (ß2-AR) triggered by catecholamine caused "targeting failure" of trastuzumab in gastric cancer cells. The antitumor activities of trastuzumab were significantly impeded by chronic catecholamine stimulation in gastric cancer cells and in the mice bearing human gastric cancer xenografts. Mechanistically, catecholamine induced upregulation of the MUC4 expression at both transcription and protein levels via activating STAT3 and ERK. The effects of catecholamine could be effectively blocked by ß2-AR antagonist ICI-118,551, indicating that ß2-AR-mediated signaling pathway plays a key role in upregulation of MUC4, which was previously demonstrated to interfere with the recognition and physical binding of trastuzumab to Her2 molecules. Moreover, a significant elevation of the MUC4 level was observed in the xenograft tissues in nude mice chronically treated with isoproterenol. Knockdown of MUC4 restored the binding activities of trastuzumab to Her2-overexpressing gastric cancer cells. In addition, coexpression of ß2-AR and MUC4 were observed in gastric cancer tissues. Our data indicated a novel trastuzumab resistance mechanism, by which catecholamine-induced ß2-AR activation mediates desensitization of gastric cancer cells to trastuzumab through upregulating the MUC4 expression.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Catecolaminas/farmacología , Resistencia a Antineoplásicos/genética , Mucina 4/genética , Receptores Adrenérgicos beta 2/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Ratones , Unión Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores Adrenérgicos beta 2/genética , Factor de Transcripción STAT3/metabolismo , Transcripción Genética/efectos de los fármacos , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7-MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.
Asunto(s)
Arsenitos/toxicidad , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/metabolismo , Ubiquitinación , Apoptosis , Línea Celular , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Estabilidad Proteica , Proteolisis/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Three fluorescent probes TP13 for thiols were rationally designed and synthesized to distinguish cysteine (Cys) from glutathione (GSH)/homocysteine (Hcy). TP13 are almost non-fluorescent and colorless 4-nitro-1,8-naphthalimide derivatives. Upon the substitution of nitro by Cys, TP13 were transformed into weakly fluorescent green-emitting 4-amino analogs via highly fluorescent blue-emitting thioether intermediates. The three-channel signaling capability allows discrimination between Cys and GSH/Hcy. The fluorescence intensity at 498 nm was linearly proportional to GSH concentration in the range of 0-20 µM, and the detection limit was 5 × 10(-8) mol L(-1). A good linear relationship between A446/A350 and Cys concentration was found in the range of 0-70 µM, and the detection limit was 2 × 10(-7) mol L(-1). Moreover, TP3 was used for living cell imaging as well as for detecting mercapto-containing proteins.
Asunto(s)
Cisteína/análisis , Colorantes Fluorescentes/química , Glutatión/análisis , Homocisteína/análisis , Línea Celular , Humanos , Límite de Detección , Imagen Óptica/métodos , Espectrometría de Fluorescencia/métodosRESUMEN
Exposure to ultraviolet B (UVB) irradiation from sunlight induces the upregulation of VEGF, a potent angiogenic factor that is critical for mediating angiogenesis-associated photodamage. However, the molecular mechanisms related to UVB-induced VEGF expression have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IκB kinase complex (IKK), IKKα, plays a critical role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs), which requires IKKα kinase activity but is independent of IKKß, IKKγ and the transactivation of NF-κB. We further show that the transcriptional factor AP-1 functions as the downstream target of IKKα that is responsible for VEGF induction under UVB exposure. Both the accumulation of AP-1 component, c-Fos and the transactivation of AP-1 by UVB require the activated IKKα located within the nucleus. Moreover, nuclear IKKα can associate with c-Fos and recruit to the vegf promoter regions containing AP-1-responsive element and then trigger phosphorylation of the promoter-bound histone H3. Thus, our results have revealed a novel independent role for IKKα in controlling VEGF expression during the cellular UVB response by regulating the induction of the AP-1 component and phosphorylating histone H3 to facilitate AP-1 transactivation. Targeting IKKα shows promise for the prevention of UVB-induced angiogenesis and the associated photodamage.
Asunto(s)
Quinasa I-kappa B/fisiología , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional , Rayos Ultravioleta , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Histonas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Quinasa I-kappa B/genética , Ratones , FN-kappa B/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Arsenite is a cytotoxic reagent that has been used clinically to treat certain cancers. Although the cytotoxic mechanisms of arsenite have been investigated, the cellular mechanisms that act against arsenite damage are poorly understood. Heme oxygenase 1 (HO-1) has been implicated in cellular survival under other multiple stress conditions. Here, we show that a significant induction of HO-1 expression is present in human bronchial epithelial cells (Beas-2B) treated with lethal doses of arsenite treatment. This induction depends on the known ERK/AP1 signaling pathway. As expected, HO-1 RNAi knockdown, or ERK/AP1 inhibition, renders the Beas-2B cells more sensitive to arsenite damage. Our data thus suggest that transcriptional upregulation of HO-1 expression via a putative ERK/AP-1 pathway constitutes an inherent mechanism by which arsenite-induced apoptosis is attenuated.
Asunto(s)
Arsenitos/toxicidad , Bronquios/citología , Células Epiteliales/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Factor de Transcripción AP-1/metabolismo , Apoptosis/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferencia de ARN , Transducción de SeñalRESUMEN
At present, a clear dependency of the dynamics upon temperature and pH has not been established for many heavy metals (HMs), so making it difficult to project and quantify the impact of ocean warming and acidification on metal biogeochemistry in future scenarios. To understand the responses of HMs to future ocean warming and acidification, we estimated the spatial-temporal variations and pollution status of six dissolved HMs (i.e., Cu, Zn, Pb, Cd, Hg, and As) in surface seawater throughout the Bohai Sea during 2012-2014. The results showed that the average concentrations of Cu, Zn, Pb, Cd, Hg, and As in seawater of the Bohai Sea were between 2.01-3.18, 10.47-15.58, 0.85-2.31, 0.25-0.55, 0.05-0.13, and 1.24-1.98 µg L-1, respectively. Spatially, the average concentrations of the studied HMs generally decreased from the three bays towards the central area, except for Hg which was relatively high in the central Bohai Sea in some cases. This implied that, in addition to continental inputs, there may be other processes affecting the distribution pattern of Hg, such as cyclonic or anticyclonic gyres, benthic fluxes between surface and bottom layers, and some marine planktonic and microbial activities. The pollution assessments of six HMs in seawater revealed that the major risk pollutants were Pb and Hg across the Bohai Sea. Analyses of the local and interactive effects of temperature and pH on HMs showed that the interactive effect of changing temperature and pH on HMs is much more complex than a direct temperature/pH relationship with HMs. Altogether, the results suggested that future ocean warming and acidification will significantly influence the concentrations of dissolved HMs in seawater of the Bohai Sea, but with different relationships.