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1.
Proc Natl Acad Sci U S A ; 119(33): e2208522119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939714

RESUMO

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.


Assuntos
Linfócitos T CD8-Positivos , Citotoxicidade Imunológica , Apoptose/fisiologia , Cromatina , Proteína Ligante Fas/metabolismo , Granzimas/genética , Humanos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos , Fatores de Transcrição
2.
Commun Biol ; 5(1): 797, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941180

RESUMO

While major changes in cellular morphology during apoptosis have been well described, the subcellular changes in nuclear architecture involved in this process remain poorly understood. Imaging of nucleosomes in cortical neurons in vitro before and during apoptosis revealed that chromatin compaction precedes the activation of caspase-3 and nucleus shrinkage. While this early chromatin compaction remained unaffected by pharmacological blockade of the final execution of apoptosis through caspase-3 inhibition, interfering with the chromatin dynamics by modulation of actomyosin activity prevented apoptosis, but resulted in necrotic-like cell death instead. With super-resolution imaging at different phases of apoptosis in vitro and in vivo, we demonstrate that chromatin compaction occurs progressively and can be classified into five stages. In conclusion, we show that compaction of chromatin in the neuronal nucleus precedes apoptosis execution. These early changes in chromatin structure critically affect apoptotic cell death and are not part of the final execution of the apoptotic process in developing cortical neurons.


Assuntos
Caspases , Cromatina , Apoptose/fisiologia , Caspase 3 , Caspases/metabolismo , Neurônios/fisiologia
3.
Cell Mol Life Sci ; 79(8): 468, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35930205

RESUMO

The mechanisms that control the inflammatory-immune response play a key role in tissue remodelling in cardiovascular diseases. T cell activation receptor CD69 binds to oxidized low-density lipoprotein (oxLDL), inducing the expression of anti-inflammatory NR4A nuclear receptors and modulating inflammation in atherosclerosis. To understand the downstream T cell responses triggered by the CD69-oxLDL binding, we incubated CD69-expressing Jurkat T cells with oxLDL. RNA sequencing revealed a differential gene expression profile dependent on the presence of CD69 and the degree of LDL oxidation. CD69-oxLDL binding induced the expression of NR4A receptors (NR4A1 and NR4A3), but also of PD-1. These results were confirmed using oxLDL and a monoclonal antibody against CD69 in CD69-expressing Jurkat and primary CD4 + lymphocytes. CD69-mediated induction of PD-1 and NR4A3 was dependent on NFAT activation. Silencing NR4A3 slightly increased PD-1 levels, suggesting a potential regulation of PD-1 by this receptor. Moreover, expression of PD-1, CD69 and NR4A3 was increased in human arteries with chronic inflammation compared to healthy controls, with a strong correlation between PD-1 and CD69 mRNA expression (r = 0.655 P < 0.0001). Moreover, PD-1 was expressed in areas enriched in CD3 infiltrating T cells. Our results underscore a novel mechanism of PD-1 induction independent of TCR signalling that might contribute to the role of CD69 in the modulation of inflammation and vascular remodelling in cardiovascular diseases.


Assuntos
Doenças Cardiovasculares , Receptor de Morte Celular Programada 1 , Apoptose/fisiologia , Humanos , Inflamação/genética , Ligantes , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos de Linfócitos T
4.
Exp Mol Med ; 54(7): 1011-1023, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35864308

RESUMO

Neutrophils are the earliest master inflammatory regulator cells recruited to target tissues after direct infection or injury. Although inflammatory factors are present in muscle that has been indirectly disturbed by peripheral nerve injury, whether neutrophils are present and play a role in the associated inflammatory process remains unclear. Here, intravital imaging analysis using spinning-disk confocal intravital microscopy was employed to dynamically identify neutrophils in denervated muscle. Slice digital scanning and 3D-view reconstruction analyses demonstrated that neutrophils escape from vessels and migrate into denervated muscle tissue. Analyses using reactive oxygen species (ROS) inhibitors and flow cytometry demonstrated that enhanced ROS activate neutrophils after denervation. Transcriptome analysis revealed that the vast majority of neutrophils in denervated muscle were of the CXCR2 subtype and were recruited by CXCL1. Most of these cells gradually disappeared within 1 week via P53-mediated apoptosis. Experiments using specific blockers confirmed that neutrophils slow the process of denervated muscle atrophy. Collectively, these results indicate that activated neutrophils are recruited via chemotaxis to muscle tissue that has been indirectly damaged by denervation, where they function in delaying atrophy.


Assuntos
Denervação Muscular , Proteína Supressora de Tumor p53 , Apoptose/fisiologia , Quimiocina CXCL1 , Humanos , Músculo Esquelético/metabolismo , Atrofia Muscular/patologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Front Cell Infect Microbiol ; 12: 889290, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35873156

RESUMO

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.


Assuntos
Neutrófilos , Tularemia , Apoptose/fisiologia , Francisella tularensis , Humanos , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tularemia/microbiologia
6.
Int J Mol Sci ; 23(15)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35897778

RESUMO

Apoptosis plays an important role in development and in the maintenance of homeostasis. Apoptotic bodies (ApoBDs) are specifically generated from apoptotic cells and can contain a large variety of biological molecules, which are of great significance in intercellular communications and the regulation of phagocytes. Emerging evidence in recent years has shown that ApoBDs are essential for maintaining homeostasis, including systemic bone density and immune regulation as well as tissue regeneration. Moreover, studies have revealed the therapeutic effects of ApoBDs on systemic diseases, including cancer, atherosclerosis, diabetes, hepatic fibrosis, and wound healing, which can be used to treat potential targets. This review summarizes current research on the generation, application, and reconstruction of ApoBDs regarding their functions in cellular regulation and on systemic diseases, providing strong evidence and therapeutic strategies for further insights into related diseases.


Assuntos
Aterosclerose , Vesículas Extracelulares , Apoptose/fisiologia , Aterosclerose/tratamento farmacológico , Comunicação Celular , Vesículas Extracelulares/fisiologia , Humanos , Fagócitos
7.
Oxid Med Cell Longev ; 2022: 1296816, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35855866

RESUMO

Ischemic stroke is a common disease that led to high mortality and high disability. NADPH oxidase 2- (NOX2-) mediated oxidative stress and long noncoding RNA have important roles in cerebral ischemia/reperfusion (CI/R) injury, whereas whether there is interplay between them remains to be clarified. This study was performed to observe the role of lncRNA PINK1-antisense RNA (PINK1-AS) in NOX2 expression regulation. An in vivo rat model (MCAO) and an in vitro cell model (H/R: hypoxia/reoxygenation) were utilized for CI/R oxidative stress injury investigation. The expression levels of lncRNA PINK1-AS, activating transcription factor 2 (ATF2), NOX2, and caspase-3 and the production level of ROS and cell apoptosis were significantly increased in CI/R injury model rats or in H/R-induced SH-SY5Y cells, but miR-203 was significantly downregulated. There was positive correlation between PINK1-AS expression level and ROS production level. PINK1-AS and ATF2 were found to be putative targets of miR-203. Knockdown of lncRNA PINK1-AS or ATF2 or the overexpression of miR-203 significantly reduced oxidative stress injury via inhibition of NOX2. Overexpression of lncRNA PINK1 significantly led to oxidative stress injury in SH-SY5Y cells through downregulating miR-203 and upregulating ATF2 and NOX2. lncRNA PINK1-AS and ATF2 were the targets of miR-203, and the lncRNA PINK1-AS/miR-203/ATF2/NOX2 axis plays pivotal roles in CI/R injury. Therefore, lncRNA PINK1-AS is a possible target for CR/I injury therapy by sponging miR-203.


Assuntos
Fator 2 Ativador da Transcrição , Isquemia Encefálica , MicroRNAs , RNA Longo não Codificante , Traumatismo por Reperfusão , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Apoptose/fisiologia , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estresse Oxidativo/genética , Proteínas Quinases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo
8.
Cell Mol Life Sci ; 79(8): 442, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35864342

RESUMO

Clostridioides difficile infection (CDI) causes nosocomial/antibiotic-associated gastrointestinal diseases with dramatically increasing global incidence and mortality rates. The main C. difficile virulence factors, toxins A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We demonstrated that TcdB induces caspase-dependent, mitochondria-independent enteric glial cell (EGC) apoptosis that is enhanced by the pro-inflammatory cytokines TNF-α and IFN-γ (CKs) by increasing caspase-3/7/9 and PARP activation. Because this cytotoxic synergism is important for CDI pathogenesis, we investigated the apoptotic pathways involved in TcdB- and TcdB + CK-induced apoptosis indepth. EGCs were pre-treated with the inhibitors BAF or Q-VD-OPh (pan-caspase), Z-DEVD-fmk (caspase-3/7), Z-IETD-fmk (caspase-8), PD150606 (calpains), and CA-074Me (cathepsin B) 1 h before TcdB exposure, while CKs were given 1.5 h after TcdB exposure, and assays were performed at 24 h. TcdB and TcdB + CKs induced apoptosis through three signalling pathways activated by calpains, caspases and cathepsins, which all are involved both in induction and execution apoptotic signalling under both conditions but to different degrees in TcdB and TcdB + CKs especially as regards to signal transduction mediated by these proteases towards downstream effects (apoptosis). Calpain activation by Ca2+ influx is the first pro-apoptotic event in TcdB- and TcdB + CK-induced EGC apoptosis and causes caspase-3, caspase-7 and PARP activation. PARP is also directly activated by calpains which are responsible of about 75% of apoptosis in TcdB and 62% in TcdB + CK which is both effector caspase-dependent and -independent. Initiator caspase-8 activation mediated by TcdB contributes to caspase-3/caspase-7 and PARP activation and is responsible of about 28% of apoptosis in both conditions. Caspase-3/caspase-7 activation is weakly responsible of apoptosis, indeed we found that it mediates 27% of apoptosis only in TcdB. Cathepsin B contributes to triggering pro-apoptotic signal and is responsible in both conditions of about 35% of apoptosis by a caspase-independent manner, and seems to regulate the caspase-3 and caspase-7 cleaved fragment levels, highlighting the complex interaction between these cysteine protease families activated during TcdB-induced apoptosis. Further a relevant difference between TcdB- and TcdB + CK-induced apoptosis is that TcdB-induced apoptosis increased slowly reaching at 72 h the value of 18.7%, while TcdB + CK-induced apoptosis increased strongly reaching at 72 h the value of 60.6%. Apoptotic signalling activation by TcdB + CKs is enriched by TNF-α-induced NF-κB signalling, inhibition of JNK activation and activation of AKT. In conclusion, the ability of C. difficile to activate three apoptotic pathways represents an important strategy to overcome resistance against its cytotoxic activity.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Apoptose/fisiologia , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Calpaína/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Caspase 7/farmacologia , Caspases/metabolismo , Catepsina B/metabolismo , Citocinas/metabolismo , Humanos , Neuroglia/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
9.
Int J Mol Sci ; 23(13)2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35806349

RESUMO

Staufen 1 (STAU1) is an RNA-binding protein that is essential in untransformed cells. In cancer cells, it is rather STAU1 overexpression that impairs cell proliferation. In this paper, we show that a modest increase in STAU1 expression in cancer cells triggers apoptosis as early as 12 h post-transfection and impairs proliferation in non-apoptotic cells for several days. Interestingly, a mutation that mimics the phosphorylation of STAU1 serine 20 is sufficient to cause these phenotypes, indicating that serine 20 is at the heart of the molecular mechanism leading to apoptosis. Mechanistically, phosphomimicry on serine 20 alters the ability of STAU1 to regulate translation and the decay of STAU1-bound mRNAs, indicating that the posttranscriptional regulation of mRNAs by STAU1 controls the balance between proliferation and apoptosis. Unexpectedly, the expression of RBD2S20D, the N-terminal 88 amino acids with no RNA-binding activity, is sufficient to induce apoptosis via alteration, in trans, of the posttranscriptional functions of endogenous STAU1. These results suggest that STAU1 is a sensor that controls the balance between cell proliferation and apoptosis, and, therefore, may be considered as a novel therapeutic target against cancer.


Assuntos
Proteínas do Citoesqueleto , Proteínas de Ligação a RNA , Serina , Apoptose/fisiologia , Transformação Celular Neoplásica , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serina/metabolismo
10.
Oxid Med Cell Longev ; 2022: 2912276, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35795857

RESUMO

Background: Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods: Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1ß was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results: Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1ß stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1ß-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion: Smad7 is highly expressed in human degenerative discs and participates in IL-1ß-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.


Assuntos
Anel Fibroso , Interleucina-1beta , Degeneração do Disco Intervertebral , Proteína Smad7 , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Apoptose/fisiologia , Caspase 9/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteína Smad7/biossíntese , Proteína Smad7/genética , Proteína Smad7/metabolismo
11.
FASEB J ; 36(8): e22449, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35839070

RESUMO

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.


Assuntos
Apoptose , Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Fatores Inibidores da Migração de Macrófagos , Neoplasias Pancreáticas , Animais , Apoptose/genética , Apoptose/fisiologia , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral/metabolismo , Movimento Celular , Proliferação de Células , Humanos , Interferons/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Eur J Histochem ; 66(3)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35855629

RESUMO

Thrombomodulin (TM) is involved in the pathological process of atherosclerosis; however, the underlying mechanism remains unclear. Oxidised low-density lipoprotein (Ox-LDL; 100 µg/mL) was used to induce human vascular smooth muscle cells (HVSMCs) into a stable atherosclerotic cell model. The expression levels of miR-550a-3p and TM were detected by real-time reverse transcription-polymerase chain reaction. Cell proliferation was estimated using CCK8 and EDU assays. Wound scratch and transwell assays were used to measure the ability of cells to invade and migrate. Propidium iodide fluorescence-activated cell sorting was used to detect apoptosis and cell cycle changes. A dual-luciferase reporter assay was performed to determine the binding of miR-550a-3p to TM. Our results suggested the successful development of a cellular atherosclerosis model. Our data revealed that TM overexpression significantly promoted the proliferation, invasion, migration, and apoptosis of HVSMCs as well as cell cycle changes. Upregulation of miR-550a-3p inhibited the growth and metastasis of HVSMCs. Furthermore, miR-550a-3p was confirmed to be a direct target of TM. Restoration of miR-550a-3p expression rescued the effects of TM overexpression. Thus, miR-550a-3p might play a role in atherosclerosis and, for the first time, normalised the function of injured vascular endothelial cells by simultaneous transfection of TM and miR-550a-3p. These results suggest that the miR-550a-3p/TM axis is a potential therapeutic target for atherosclerosis.


Assuntos
Aterosclerose , MicroRNAs , Apoptose/fisiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Proliferação de Células/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Trombomodulina/genética , Trombomodulina/metabolismo
13.
Oxid Med Cell Longev ; 2022: 8279269, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35903712

RESUMO

Hair follicles (HFs) maintain homeostasis through the hair cycles; therefore, disrupting the hair cycle may lead to hair loss. Our previous study showed that apoptosis-inducing factor (AIF) nuclear translocation and poly [ADP-ribose] polymerase 1 (PARP1) upregulation induced apoptosis in mouse hair follicles during the hair cycle transition from anagen to catagen. However, the mechanism underlying this phenomenon remains unclear. In this study, we found that intrinsic ROS levels increased during the hair follicle cycle transition from anagen to catagen, followed by abrupt DNA breaks and activation of homologous recombinant and nonhomologous end joining DNA repair, along with the enhancement of apoptosis. Mice in different stages of the hair cycle were sacrificed, and the dorsal skins were collected. The results of western blot and histological staining indicated that AIF-PARP1 plays a key role in HF apoptosis, but their role in the regulation of the HF cycle is not clear. Mice were treated with inhibitors from anagen to catagen: treatment with BMN 673, a PARP1 inhibitor, increased DNA breaks and activated the cytochrome c/caspase-3-mediated apoptotic pathway, accelerating HF regression. Ac-DEVD-CHO (Ac), a caspase-3 inhibitor, attenuated HF degeneration by upregulating PARP1 expression, suggesting a seesaw relationship between cytochrome c-caspase-3- and AIF-PARP1-mediated apoptosis, wherein PARP1 may be the fulcrum. In addition, macrophages were involved in regulating the hair cycle, and the rate of M1 macrophages around HFs increased during catagen, while more M2 macrophages were found during anagen and telogen. Our results indicate that intrinsic ROS drive HF cycle progression through DNA damage and repair, followed by apoptosis. Intrinsic ROS drive hair follicle cycle progression by modulating DNA damage and repair, and consecutively, hair follicle apoptosis and macrophage polarization work together to promote the hair follicle cycle.


Assuntos
Citocromos c , Folículo Piloso , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Caspase 3/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Dano ao DNA , Macrófagos/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo
14.
Drug Deliv ; 29(1): 2498-2512, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35903814

RESUMO

Spinal cord injury (SCI) is a serious central nervous system disease, and secondary injury, including oxidative stress, the inflammatory response and accompanying neuronal apoptosis, will aggravate the condition. Due to the existence of the blood-spinal cord barrier (BSCB), the existing drugs for SCI treatment are difficulty to reach the injury site and thus their efficacy is limited. In this study, we designed chitosan-modified hollow manganese dioxide nanoparticles (CM) for the delivery of resveratrol to help it pass through the BSCB. Resveratrol (Res), a poorly soluble drug, was adsorbed into CM with a particle size of approximately 130 nm via the adsorption method, and the drug loading reached 21.39 ± 2.53%. In vitro dissolution experiment, the Res release of the loaded sample (CMR) showed slowly release behavior and reached about 87% at 36 h. In vitro at the cellular level and in vivo at the animal level experiments demonstrated that CMR could alleviate significantly oxidative stress by reducing level of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and increasing glutathione peroxidase (GSH) level. Additionally, immunofluorescence (iNOS, IL-1ß, and Cl caspase-3) and western blot (iNOS, cox-2, IL-1ß, IL-10, Cl caspase-3, bax, and bcl-2) were used to detect the expression of related factors, which verified that CMR could also reduce inflammation and neuronal apoptosis. These results indicated that CM, as a potential central nervous system drug delivery material, was suitable for SCI treatment.


Assuntos
Quitosana , Nanopartículas , Traumatismos da Medula Espinal , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Quitosana/metabolismo , Compostos de Manganês , Estresse Oxidativo , Óxidos , Ratos , Ratos Sprague-Dawley , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico
15.
Int J Mol Med ; 50(3)2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35775376

RESUMO

Bladder cancer is the most common malignant tumor of the urinary system, and in China it is first among urogenital system tumors. More therapeutic targets are still urgently required to combat this disease. Lamin B2 (LMNB2) is a type of nuclear lamina filament protein, which is involved in multiple cellular processes, and known as an oncogene affecting the progression of multiple types of cancers. Although the multiple effects of LMNB2 on cancer progression have been elucidated, its possible role in bladder cancer remains unclear. In the present study, it was determined that LMNB2 expression was upregulated in human bladder cancer tissues, and its expression was correlated with the prognosis and the clinical features, including tumor stage (P=0.001) and recurrence (P=0.006) of patients with bladder cancer. In addition, it was further revealed that LMNB2 depletion inhibited bladder cancer cell proliferation, stimulated cell cycle arrest and apoptosis in vitro, and suppressed tumor growth of bladder cancer cells in mice. Furthermore, the present data revealed that LMNB2 promoted the proliferation of bladder cancer cells via transcriptional activation of CDCA3 expression. Therefore, the role of LMNB2 in bladder cancer progression was demonstrated, and may serve as a promising therapeutic target for bladder cancer treatment.


Assuntos
Lamina Tipo B , Neoplasias da Bexiga Urinária , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Lamina Tipo B/biossíntese , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Camundongos , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
16.
Cells ; 11(12)2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35741016

RESUMO

Members of the caspase family are well known for their roles in the initiation and execution of cell death. Due to their function in the removal of damaged cells that could otherwise become malignant, caspases are important players in the DNA damage response (DDR), a network of pathways that prevent genomic instability. However, emerging evidence of caspases positively or negatively impacting the accumulation of DNA damage in the absence of cell death demonstrates that caspases play a role in the DDR that is independent of their role in apoptosis. This review highlights the apoptotic and non-apoptotic roles of caspases in the DDR and how they can impact genomic stability and cancer treatment.


Assuntos
Apoptose , Caspases , Apoptose/fisiologia , Caspases/metabolismo , Morte Celular , Dano ao DNA , Instabilidade Genômica , Humanos
17.
Cells ; 11(12)2022 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-35741037

RESUMO

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.


Assuntos
Apoptose , Receptor fas , Apoptose/fisiologia , Análise por Conglomerados , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor fas/metabolismo
18.
Molecules ; 27(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35684320

RESUMO

TIR-domain-containing adapter-inducing interferon-ß (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.


Assuntos
Amiloide , Necroptose , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Amiloide/metabolismo , Apoptose/fisiologia
19.
Front Immunol ; 13: 836754, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35693822

RESUMO

Neutrophils (polymorphonuclear leukocytes, PMNs) have a distinctively short lifespan, and tight regulation of cell survival and death is imperative for their normal function. We demonstrated previously that Francisella tularensis extends human neutrophil lifespan, which elicits an impaired immune response characterized by neutrophil dysfunction. Herein, we extended these studies, including our transcriptional profiling data, and employed Seahorse extracellular flux analysis, gas chromatography-mass spectrometry metabolite analysis, flow cytometry and several other biochemical approaches to demonstrate that the delayed apoptosis observed in F. tularensis-infected neutrophils is mediated, in part, by metabolic reprogramming. Specifically, we show that F. tularensis-infected neutrophils exhibited a unique metabolic signature characterized by increased glycolysis, glycolytic flux and glucose uptake, downregulation of the pentose phosphate pathway, and complex glycogen dynamics. Glucose uptake and glycolysis were essential for cell longevity, although glucose-6-phosphate translocation into the endoplasmic reticulum was not, and we identify depletion of glycogen as a potential trigger of apoptosis onset. In keeping with this, we also demonstrate that ablation of apoptosis with the pan-caspase inhibitor Q-VD-OPh was sufficient to profoundly increase glycolysis and glycogen stores in the absence of infection. Taken together, our data significantly advance understanding of neutrophil immunometabolism and its capacity to regulate cell lifespan.


Assuntos
Francisella tularensis , Tularemia , Apoptose/fisiologia , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Neutrófilos
20.
PLoS Genet ; 18(6): e1010232, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35727824

RESUMO

Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.


Assuntos
Autofagossomos , Dineínas do Citoplasma , Células Ciliadas Auditivas , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Cóclea/citologia , Cóclea/metabolismo , Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Camundongos
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