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Ferroptosis, a type of regulated cell death brought about by lipid peroxidation, has been discovered to suppress tumor growth. Here, we report that targeting RRM1 promotes ferroptosis and affects sensitivity to radiation and chemotherapeutics in cancer cells. In vitro experiments demonstrate that RRM1 increases the accumulation of cellular reactive oxygen species (ROS) and lipid peroxidation by disrupting the activity and expression of the antioxidant enzyme GPX4. Further studies reveal the downstream mechanisms of RRM1, which can regulate the deubiquitinating enzyme USP11 and ubiquitinating enzyme MDM2 to affect the ubiquitination modification of p53. Unstable p53 then inhibited the activity and expression of GPX4 by restraining the p21 protein. Furthermore, our data reveal that targeting RRM1 also increases radiation-induced DNA damage and apoptotic signaling and causes crosstalk between ferroptosis and apoptosis. On the basis of our collective findings, we propose that RRM1 is an essential negative mediator of radiosensitivity through regulating ferroptosis, which could serve as a potential target to inhibit the tumor's antioxidant system and enhance the efficiency of radio/chemotherapy.
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HIGD1A is an important mitochondrial protein recently shown to have a novel nuclear localization under severe stress. However, whether this protein is also associated with the DNA damage response has rarely been studied. Here, we reported that DSBs-induced the translocation of mitochondrial HIGD1A to the nucleus is dependent on nuclear pore complex (NPCs), which finally promotes HR and radio/chemo-resistance. Importantly, NUP93 and HIGD1A physically interact and the interaction domain with NUP93 is located at residues 46-60 of HIGD1A. Chromatin-enriched HIGD1A can then directly interact with RPA. During the early stages of HR, HIGD1A promotes the loading of RPA to DSBs and activates the DNA damage-dependent chromatin association of RAD9-RAD1-HUS1 complex (9-1-1), which stimulates the ATR-Chk1-dependent G2/M DNA damage checkpoint. After facilitating RPA-ssDNA binding, HIGD1A in turn inhibits abnormal persistence of RPA1 foci by promoting ubiquitination of RPA1 and inducing its eventual proteasomal degradation. In addition, we have identified clinical drug Preveon associated with the HIGD1A-NUP93 interaction domain using a virtual screening approach. This compound directly interacted with HIGD1A, which was verified by NMR, and then inhibited HIGD1A translocation. Collectively, we demonstrate a novel role for HIGD1A in DSBs and provide rationale for using HIGD1A inhibitors as cancer therapeutics.
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Proteínas de Ciclo Celular , Dano ao DNA , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Recombinação Homóloga , HumanosRESUMO
BACKGROUND: Muscle-invasive bladder cancer (MIBC) patients are subject to unfavorable treatment options and a high recurrence rate. The status of TP53 mutations played an essential role in the progression and the prognosis of MIBC. The present study proposed to investigate the association between TP53 mutations and immunophenotype in MIBC. RESULTS: We established an immune prognostic model (IPM) ground on the immune-associated genes derived from variation analysis between wild-type TP53 and mutated TP53 TCGA-MIBC patients, and validated in another cohort from GEO database. Based on IPM, we divided MIBC patients into low and high risk subgroups. The high risk MIBC patients had higher proportions of macrophages M1, and lower proportions of T cells regulatory (Tregs) and activated dendritic cells than the low risk MIBC patients. Moreover, the expression of immune checkpoints genes (PD1, CTLA4, LAG3, HAVCR2 and TIGIT) was higher in the high risk patients than the low risk patients. In clinical application, IPM exhibited better survival prediction than conventional clinical characteristics. CONCLUSIONS: Our investigation presented practical prognostic significance for MIBC patients and displayed the overarching landscape of the immune response in the MIBC microenvironment. METHODS: Data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) analysis between the TP53 mutated and wild-type MIBC patients was conducted. The CIBERSORT algorithm was performed to evaluate the proportion of immune cell types. Gene expression profiles from the TCGA and GEO were used as training and testing cohorts to build and validate an immune-related prognostic model (IPM). Genes in the IPM model were first screened by univariate Cox analysis, then filtered by the least absolute shrinkage and selection operator (LASSO) Cox regression. A nomogram was finally established and evaluated by combining both the IPM and other clinical factors.
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Carcinoma de Células de Transição/genética , Músculo Liso/patologia , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Carcinoma de Células de Transição/imunologia , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Bases de Dados Genéticas , Células Dendríticas/imunologia , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Estimativa de Kaplan-Meier , Macrófagos/imunologia , Mutação , Invasividade Neoplásica , Nomogramas , Prognóstico , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Modelos de Riscos Proporcionais , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Reprodutibilidade dos Testes , Taxa de Sobrevida , Linfócitos T Reguladores/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
BACKGROUND: Retinal degeneration is a leading cause of blindness in the world; its etiology is complex and involves genetic defects and stress-associated aging. In addition to gene therapies for known genetically defective retinal degeneration, cellular therapies have been widely explored for restoring vision in both preclinical animal models and clinical trials. Stem cells of distinct tissue sources and their derived lineages have been tested for treating retinal degeneration; most of them were reported to be effective to some extent in restoring/improving deteriorated vision. Whether this visual improvement is due to a functional integration of grafted cells to substitute for lost retinal neurons in recipients or due to their neuroprotective and neurotrophic effects to retain recipient functional neurons, or both, is still under debate. METHODS: We compared the results of subretinal transplantation of various somatic cell types, such as stem cells and differentiated cells, into RhoP23H/+ mice, a retinal degeneration model for human retinitis pigmentosa (RP) by evaluating their optokinetic response (OKR) and retinal histology. We identified some paracrine factors in the media that cultured cells secreted by western blotting (WB) and functionally evaluated the vascular endothelial growth factor Vegfa for its potential neurotrophic and neuroprotective effects on the neuroretina of model animals by intravitreal injection of VEGF antibody. RESULTS: We found that live cells, regardless of whether they were stem cells or differentiated cell types, had a positive effect on improving degenerating retinas after subretinal transplantation; the efficacy depended on their survival duration in the host tissue. A few paracrine factors were identified in cell culture media; Vegfa was the most relevant neurotrophic and neuroprotective factor identified by our experiments to extend neuron survival duration in vivo. CONCLUSIONS: Cellular therapy-produced benefits for remediating retinal degeneration are mostly, if not completely, due to a paracrine effect of implanted cells on the remaining host retinal neurons.
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Degeneração Retiniana , Retinose Pigmentar , Animais , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Retina , Degeneração Retiniana/terapia , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Fator A de Crescimento do Endotélio VascularRESUMO
Response to radiotherapy (RT) in cancers varies widely among patients. Therefore, it is very important to predict who will benefit from RT before clinical treatment. Consideration of the immune tumor microenvironment (TME) could provide novel insight into tumor treatment options. In this study, we investigated the link between immune infiltration status and clinical RT outcome in order to identify certain leukocyte subsets that could potentially influence the clinical RT benefit across cancers. By integrally analyzing the TCGA data across seven cancers, we identified complex associations between immune infiltration and patients RT outcomes. Besides, immune cells showed large differences in their populations in various cancers, and the most abundant cells were resting memory CD4 T cells. Additionally, the proportion of activated CD4 memory T cells and activated mast cells, albeit at low number, were closely related to RT overall survival in multiple cancers. Furthermore, a prognostic model for RT outcomes was established with good performance based on the immune infiltration status. Summarized, immune infiltration was found to be of significant clinical relevance to RT outcomes. These findings may help to shed light on the impact of tumor-associated immune cell infiltration on cancer RT outcomes, and identify biomarkers and therapeutic targets.
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BACKGROUND: The retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols. METHODS: Sphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches. FINDINGS: We provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming. INTERPRETATION: The Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. FUND: National Natural Science Foundation of China and the National Institute of Health USA.
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Reprogramação Celular , Epitélio Pigmentado da Retina/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Biomarcadores , Desdiferenciação Celular/genética , Diferenciação Celular/genética , Movimento Celular , Separação Celular/métodos , Células Cultivadas , Senescência Celular/genética , Epigênese Genética , Imunofluorescência , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Transgênicos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Transdução de Sinais , Testes VisuaisRESUMO
To explore possible approaches to differentiating rat bone marrow mesenchymal stem cells (BMSCs) into retinal ganglion-like cells and to demonstrate the dynamic changes in protein expression profiles of BMSCs throughout the differentiation. BMSCs were isolated from adult rats and cultured in medium conditioned by neonatal rat retinal cells to induce BMSC differentiation into retinal ganglion-like cells. Immunostaining for neurofilament, nestin, Map2, and Thy1.1 was used to follow the differentiation process. Two types of protein arrays were employed to profile the BMSCs, the differentiated retinal ganglion-like cells, and the primary retinal ganglion cells (RGCs) using the Biomarker Wizard System. After 7 days of culture in conditioned medium, cells showing a neural-cell-like modality appeared. The differentiated retinal ganglion-like cells showed that network-like connections were positive for nestin, neurofilament, Map2, and Thy1.1. In total, 16 marker proteins were highly expressed in both retinal ganglion-like cells and RGCs and no obvious expression was observed in BMSCs. Among them, nine proteins were expressed more highly in RGCs than in retinal ganglion-like cells. BMSCs can be induced to differentiate into retinal ganglion-like cells by neonatal rat retinal cells, and the induced cells show protein profiles resembling those of isolated RGCs.
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Proteínas Associadas aos Microtúbulos/genética , Nestina/genética , Células Ganglionares da Retina/metabolismo , Antígenos Thy-1/genética , Animais , Animais Recém-Nascidos/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Filamentos Intermediários/genética , Células-Tronco Mesenquimais , Ratos , Células Ganglionares da Retina/citologia , Transcriptoma/genéticaRESUMO
Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in "initiator" caspases, "effector" caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both "initiator" and "effector" caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.
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Apoptose/efeitos da radiação , Mitocôndrias/efeitos da radiação , Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Caspase 3/genética , Caspase 7/genética , Caspase 9/genética , Morte Celular , Fibroblastos/efeitos da radiação , Células HCT116 , Humanos , Camundongos , Mitocôndrias/genética , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/efeitos da radiação , Neoplasias/patologia , Neoplasias/radioterapiaRESUMO
As a quorum-sensing molecule for bacteria-bacteria communication, N-(3-oxododecanoyl)-homoserine lactone (C12) has been found to possess pro-apoptotic activities in various cell culture models. However, the detailed mechanism of how this important signaling molecule function in the cells of live animals still remains largely unclear. In this study, we systematically investigated the mechanism for C12-mediated apoptosis and studied its anti-tumor effect in Caenorhabditis elegans (C. elegans). Our data demonstrated that C12 increased C. elegans germ cell apoptosis, by triggering mitochondrial outer membrane permeabilization (MOMP) and elevating the reactive oxygen species (ROS) level. Importantly, C12-induced ROS increased the expression of genes critical for DNA damage response (hus-1, clk-2 and cep-1) and genes involved in p38 and JNK/MAPK signaling pathway (nsy-1, sek-1, pmk-1, mkk-4 and jnk-1). Furthermore, C12 failed to induce germ cell apoptosis in animals lacking the expression of each of those genes. Finally, in a C. elegans tumor-like symptom model, C12 significantly suppressed tumor growth through inhibiting the expression of RAS/MAPK pathway genes (let-23/EGFR, let-60/RAS, lin-45/RAF, mek-2/MEK and mpk-1/MAPK). Overall, our results indicate that DNA damage response and MAPK activation triggered by mitochondrial ROS play important roles in C12-induced apoptotic signaling in C. elegans, and RAS/MAPK suppression is involved in the tumor inhibition effect of C12. This study provides in vivo evidence that C12 is a potential candidate for cancer therapeutics by exerting its pro-apoptotic and anti-tumor effects via elevating mitochondria-dependent ROS production.
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4-Butirolactona/análogos & derivados , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Células Germinativas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , 4-Butirolactona/farmacologia , Animais , Apoptose/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Carcinogênese/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Células Germinativas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mitocôndrias/metabolismo , Mutação , Estresse Oxidativo , Interferência de RNA , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
In age-related macular degeneration (AMD), abnormal sub retinal choroidal neovascularization (CNV) is a major cause of blindness. FR-sema3C is a point mutated form of semaphorin-3C that is resistant to cleavage by furin like pro-protein convertases (FPPC). We have found in previous work that FR-sema3C functions as an anti-angiogenic factor. In this study we investigated the possible use of FR-sema3C as an inhibitor of CNV. FR-sema3C inhibits VEGF as well as PDGF-BB signal transduction in endothelial cells and to less extent bFGF induced signal transduction using a mechanism that does not depend upon the binding of VEGF like the drugs that are currently the mainstay treatment for AMD. CNV was induced in eyes of C57 black mice by laser photocoagulation. Intravitreal injection of FR-Sema3C or aflibercept (VEGF-trap) was then used to inhibit CNV formation. Invading choroidal vessels were visualized a week later by injection of FITC-dextran into the circulation, followed by the measurement of the area of the invading blood vessels. Injection of 0.1 µg FR-Sema3C inhibited CNV by 55% (P<0.01) and was as effective as 5 µg aflibercept. FR-sema3C did not display any adverse effects on retinal function following its injection into eyes of healthy mice as assessed by optokinetic reflex (OKR) and Electro-retinogram (ERG) criteria. Furthermore, FR-sema3C did not induce apoptosis in the retina as determined by TUNEL nor was there any discernable structural damage to the retina as assessed by several immuno-histochemical criteria. Our results suggest that FR-sema3C could perhaps be used for the treatment of AMD, and that it may perhaps be of benefit to patients that do not respond well to current treatments relying on VEGF sequestering agents.
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Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/metabolismo , Furina/metabolismo , Furina/farmacologia , Semaforinas/metabolismo , Semaforinas/farmacologia , Inibidores da Angiogênese/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular , Dextranos/administração & dosagem , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Intravítreas , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Retina/efeitos dos fármacos , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Abnormal subretinal choroidal neovascularization (CNV) is a major cause of blindness in exudative age-related macular degeneration (AMD). Current anti-angiogenic treatments by VEGF sequestering agents have been successful, but a significant proportion of patients do not respond well to these treatments, and the response of others diminishes over time, suggesting that additional anti-angiogenic agents that function by separate mechanisms may be of use to such patients. We have previously found that a point mutated form of semaphorin-3E resistant to cleavage by furin like pro-protein convertases (UNCL-Sema3E) displays potent anti-angiogenic properties. We therefore determined if UNCL-Sema3E has potential as an inhibitor of CNV formation. We chose to study UNCL-Sema3E rather than wild type sema3E because unlike full length sema3E, the major p61-Sema3E peptide that is produced by cleavage of sema3E with furin like pro-protein convertases activates signal transduction mediated by the ErbB2 receptor and can promote tumor metastasis in addition to its anti-angiogenic activity. UNCL-Sema3E inhibited efficiently vascular endothelial growth factor-A (VEGF), platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) signaling in human umbilical vein derived endothelial cells (HUVEC) and to a lesser extent hepatocyte growth factor (HGF) signal transduction. CNV that was induced in the eyes of C57 black mice by laser photocoagulation was inhibited by 65% (P < 0.01) following a single bolus intra-vitreal injection of 5 µg UNCL-Sema3E. This inhibitory effect was similar to the inhibition produced by a single bolus intra-vitreal injection of 5 µg aflibercept. A similar inhibition of CNV was observed following the injection of UNCL-Sema3E into the eyes of Long-Evans rats. However, a higher dose of UNCL-Sema3E (125 µg), partially due to the larger volume of the vitreous cavity of rats, was required to achieve maximal inhibition of CNV. Injection of UNCL-Sema3E into eyes of healthy mice did not have any adverse effect on retinal function as assessed by optic kinetic reflex (OKR) or by electroretinogram (ERG) assays nor did UNCL-Sema3E injection affect the structure of the retina as determined using histology. To conclude, our results suggest that UNCL-Sema3E may be useful for the treatment of exudative AMD, which does not respond well to conventional anti-VEGF therapy.
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Neovascularização de Coroide/tratamento farmacológico , Glicoproteínas/administração & dosagem , Proteínas de Membrana/administração & dosagem , Mutação Puntual , Proteínas de Ligação a RNA/administração & dosagem , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Glicoproteínas/genética , Humanos , Injeções Intravítreas , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genética , Ratos , Ratos Long-Evans , SemaforinasRESUMO
Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.
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4-Butirolactona/análogos & derivados , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Homosserina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , 4-Butirolactona/farmacologia , Animais , Apoptose/efeitos dos fármacos , Arildialquilfosfatase/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Imunofluorescência , Homosserina/farmacologia , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein disulfide isomerase (PDI) family proteins are classified as enzymatic chaperones for reconstructing misfolded proteins. Previous studies have shown that several PDI members possess potential proapoptotic functions. However, the detailed molecular mechanisms of PDI-mediated apoptosis are not completely known. In this study, we investigated how two members of PDI family, PDI and PDIA3, modulate apoptotic signaling. Inhibiting PDI and PDIA3 activities pharmacologically alleviates apoptosis induced by various apoptotic stimuli. Although a decrease of PDIA3 expression alleviates apoptotic responses, overexpression of PDIA3 exacerbates apoptotic signaling. Importantly, Bak, but not Bax, is essential for PDIA3-induced proapoptotic signaling. Furthermore, both purified PDI and PDIA3 proteins induce Bak-dependent, but not Bax-dependent, mitochondrial outer membrane permeabilization in vitro, probably through triggering Bak oligomerization on mitochondria. Our results suggest that both of PDI and PDIA3 possess Bak-dependent proapoptotic function through inducing mitochondrial outer membrane permeabilization, which provides a new mechanism linking ER chaperone proteins and apoptotic signaling.
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Apoptose , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Animais , Biocatálise , Imunofluorescência , CamundongosRESUMO
Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2(-/flox) mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.
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Neovascularização de Coroide/terapia , Quinase I-kappa B/antagonistas & inibidores , Animais , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Lasers , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Transdução de SinaisRESUMO
Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.
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Inibidores da Angiogênese/metabolismo , Astrócitos/metabolismo , Neovascularização de Coroide/metabolismo , Exossomos/metabolismo , Retina/citologia , Animais , Células da Medula Óssea/citologia , Movimento Celular , Células Cultivadas , Quimiotaxia , Feminino , Inflamação , Lasers , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The Tyro3, Axl and Mertk (TAM) triply knockout (TKO) mice exhibit systemic autoimmune diseases, with characteristics of increased proinflammatory cytokine production, autoantibody deposition and autoreactive lymphocyte infiltration into a variety of tissues. Here we show that TKO mice produce high level of serum TNF-α and specific autoantibodies deposited onto brain blood vessels. The brain-blood barrier (BBB) in mutant brains exhibited increased permeability for Evans blue and fluorescent-dextran, suggesting a breakdown of the BBB in the mutant brains. Impaired BBB integrity facilitated autoreactive T cells infiltrating into all regions of the mutant brains. Brain autoimmune disorder caused accumulation of the ubiquitin-reactive aggregates in the mutant hippocampus, and early formation of autofluorescent lipofuscins in the neurons throughout the entire brains. Chronic neuroinflammation caused damage of the hippocampal mossy fibers and neuronal apoptotic death. This study shows that chronic systemic inflammation and autoimmune disorders in the TKO mice cause neuronal damage and death.
Assuntos
Apoptose , Doenças Autoimunes/genética , Dano Encefálico Crônico/imunologia , Região CA3 Hipocampal/patologia , Neurônios/fisiologia , Animais , Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Barreira Hematoencefálica/metabolismo , Dano Encefálico Crônico/genética , Dano Encefálico Crônico/patologia , Região CA3 Hipocampal/irrigação sanguínea , Região CA3 Hipocampal/imunologia , Permeabilidade Capilar/imunologia , Células Cultivadas , Citocinas/metabolismo , Giro Denteado/irrigação sanguínea , Giro Denteado/imunologia , Giro Denteado/patologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Corpos de Inclusão/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/imunologia , Microvasos/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue , Proteínas Ubiquitinadas/metabolismo , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Apoptosis is recognized as an important mechanism in contrast-induced nephropathy (CIN). As tetramethylpyrazine (TMP) has been recently found to be renoprotective and anti-apoptotic in multiple kidney injuries, we hypothesized that TMP would prevent CIN. METHODS: An experimental model of CIN was established in rats. Serum creatinine, blood urea nitrogen, plasma cystatin C, urinary N-acetyl-ß-glucosaminidase, and urinary γ-glutamyl transpeptidase were measured to evaluate kidney function. Apoptosis was assessed by transmission electron microscopy, transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and poly-ADP-ribose polymerase cleavage. Fork-head box O1 transcriptional factor (FoxO1) mRNA expression was evaluated by quantitative real-time PCR. Phospho-p38 mitogen-activated protein kinase (MAPK) protein expression was assessed by immunohistochemistry and Western blotting. RESULTS: TMP significantly attenuated the resulting renal dysfunction and renal tubular cell apo-ptosis. Mechanistically, TMP decreased the expression of phospho-p38 MAPK protein and attenuated the increased FoxO1 mRNA and nuclear protein expression. In addition, TMP inhibited inducible nitric oxide synthase and Bax protein expression while it upregulated Bcl-2. CONCLUSION: In summary, this study demonstrated the protective role of TMP against CIN and indicated the effects of TMP may be mediated by the inhibition of p38 MAPK and FoxO1 pathways. Thus, TMP may be a new potential therapeutic agent to prevent CIN.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Apoptose/efeitos dos fármacos , Meios de Contraste/efeitos adversos , Fatores de Transcrição Forkhead/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Injúria Renal Aguda/prevenção & controle , Animais , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Necrose do Córtex Renal/induzido quimicamente , Necrose do Córtex Renal/prevenção & controle , Masculino , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: The Notch signaling pathway plays crucial roles in regulation of cell proliferation, differentiation and cell fate decision in multiple tissues and cell types. This study was designed to test the effects of enhanced Notch activity on corneal epithelium homeostasis and wound healing using the transgenic mice that overexpressed an activated Notch1 (NICD) in cornea epithelium. METHODS: The studies were performed on R26(fN1-ICD) transgenic mice that carry a NICD cDNA (cDNA) whose expression is prevented by a "Lox-STOP-Lox" cassette. When this transgenic mouse is bred to a mouse strain carrying a Cre recombinase expression cassette driven by a tissue-specific keratin 14 (K14) promoter, the floxed "STOP" cassette is excised and NICD is expressed in the cornea epithelium. The expression level of NICD and its downstream target genes, hairy and enhancer of split 1 (Hes1) and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), in the transgenic corneal epithelium was examined by quantitative PCR (qPCR). The phenotypes and morphology of the transgenic corneal epithelium were compared with that of wild type (WT) controls. The proliferation rate of the epithelial cells was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the differentiation statues were examined by K14, tumor protein p63 (p63), K12, and zona occludens 1 (ZO-1) immunoreactivity at either normal developmental condition or after corneal epithelial debridement. The corneal epithelial response to wound healing was studied by fluorescent staining and Richardson's staining macroscopically and by H&E staining at microscope level at 0, 6, 12, 18, and 24 h post injury. RESULTS: Although overexpression of NICD in cornea epithelium led to upregulation of its downstream targets, i.e., Hes1 and Hey1, this did not alter corneal epithelial cell proliferation and differentiation. However, wound healing induced Notch activity and overexpression of NICD promoted corneal epithelial wound healing, which was in agreement with more rapid early proliferation response in NICD transgenic mice than in the wild type control mice. CONCLUSIONS: These findings further demonstrate the functional role of Notch signaling in corneal epithelium wound healing response.
Assuntos
Epitélio Corneano/lesões , Epitélio Corneano/fisiopatologia , Receptor Notch1/fisiologia , Cicatrização/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Primers do DNA/genética , Epitélio Corneano/patologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Homeostase , Camundongos , Camundongos Transgênicos , Receptor Notch1/genética , Transdução de Sinais , Fatores de Transcrição HES-1 , Cicatrização/genéticaRESUMO
14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using 'repeated epilation' heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.