RESUMO
Chemotherapies are standard care for most cancer types. Pyrimidine analogs including 5-fluorouracil, cytosine arabinoside, 5-azacytidine, and gemcitabine are effective drugs that are utilized as part of a number of anticancer regimens. However, their lack of cell-specificity results in severe side effects. Therefore, there is a capacity to improve the efficacy of such therapies, while decreasing unwanted side effects. Here, we report that while 5-fluorocytosine is not chemotherapeutic in itself, incorporated into a ribonucleoside and more importantly into an RNA oligonucleotide, it induces cytotoxic effects on cancer cells in vitro . Interestingly, these effects are rescued by both uridine and thymidine. Similarly, in-vitro 2'-deoxy-5-fluorocytidine inhibits the growth of tumor cells but has the advantage of being less toxic to human primary cells compared with 5-fluorocytidine, suggesting that the deoxyribonucleoside could exhibit less side-effects in vivo . Thus, this work indicates that the potency of 5-fluorocytidine and 2'-deoxy-5-fluorocytidine should be further explored. In particular, oligonucleotides incorporating 5-fluorocytosine could be novel chemotherapeutic drugs that could be formulated in cancer-specific particles for safe and efficacious cancer treatments.
RESUMO
Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1ß by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1ß generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1ß-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.
Assuntos
Caspase 1/metabolismo , Vesículas Extracelulares/metabolismo , Inflamassomos/imunologia , Macrófagos/imunologia , Peroxirredoxinas/fisiologia , Choque Séptico/prevenção & controle , Animais , Caspase 1/genética , Citocinas/metabolismo , Feminino , Inflamassomos/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Choque Séptico/patologia , Transdução de SinaisRESUMO
The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.
Assuntos
Epiderme , Sistemas Microfisiológicos , Adulto , Humanos , Diferenciação Celular , Epiderme/metabolismo , Células Epidérmicas/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo , Transglutaminases/metabolismoRESUMO
Mammalian cells export most proteins by the endoplasmic reticulum/Golgi-dependent pathway. However, some proteins are secreted via unconventional, poorly understood mechanisms. The latter include the proinflammatory cytokines interleukin(IL)-1beta, IL-18, and IL-33, which require activation by caspase-1 for biological activity. Caspase-1 itself is activated by innate immune complexes, the inflammasomes. Here we show that secretion of the leaderless proteins proIL-1alpha, caspase-1, and fibroblast growth factor (FGF)-2 depends on caspase-1 activity. Although proIL-1alpha and FGF-2 are not substrates of the protease, we demonstrated their physical interaction. Secretome analysis using iTRAQ proteomics revealed caspase-1-mediated secretion of other leaderless proteins with known or unknown extracellular functions. Strikingly, many of these proteins are involved in inflammation, cytoprotection, or tissue repair. These results provide evidence for an important role of caspase-1 in unconventional protein secretion. By this mechanism, stress-induced activation of caspase-1 directly links inflammation to cytoprotection, cell survival, and regenerative processes.
Assuntos
Caspase 1/imunologia , Caspase 1/metabolismo , Animais , Células COS , Caspase 1/genética , Linhagem Celular , Chlorocebus aethiops , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Inflamação/imunologia , Interleucina-1alfa/metabolismo , Queratinócitos/imunologia , Macrófagos/imunologia , Camundongos , Ligação Proteica , Sinais Direcionadores de Proteínas , Proteômica , RNA Interferente Pequeno/genéticaRESUMO
Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.
Assuntos
Queratinócitos , Fator 2 Relacionado a NF-E2/fisiologia , Pele , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Pele/citologia , Pele/metabolismoRESUMO
Multivessel coronary artery disease (CAD) is characterized by underlying chronic vascular inflammation and occlusion in the coronary arteries, where these patients undergo coronary artery bypass grafting (CABG). Since post-cardiotomy inflammation is a well known phenomenon after CABG, attenuation of this inflammation is required to reduce perioperative morbidity and mortality. In this study, we aimed to phenotype circulating frequencies and intensities of monocyte subsets and monocyte migration markers, respectively, and to investigate the plasma level of inflammatory cytokines and chemokines between preoperative and postoperative CAD patients and later, to intervene the inflammation with sodium selenite. We found a higher amplitude of inflammation, postoperatively, in terms of CCR1high monocytes and significantly increased pro-inflammatory cytokines, IL-6, IL-8, and IL-1RA. Further, in vitro intervention with selenium displayed mitigating effects on the IL-6/STAT-3 axis of mononuclear cells derived from postoperative CAD patients. In addition, in vitro selenium intervention significantly reduced IL-1ß production as well as decreased cleaved caspase-1 (p20) activity by preoperative (when stimulated) as well as postoperative CAD mononuclear cells. Though TNF-α exhibited a positive correlation with blood troponin levels in postoperative CAD patients, there was no obvious effect of selenium on the TNF-α/NF-κB axis. In conclusion, anti-inflammatory selenium might be utilized to impede systemic inflammatory cytokine axes to circumvent aggravating atherosclerosis and further damage to the autologous bypass grafts during the post-surgical period.
Assuntos
Doença da Artéria Coronariana , Selênio , Humanos , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/cirurgia , Citocinas/genética , Imunofenotipagem , Inflamação , Interleucina-6/farmacologia , Monócitos , Selênio/farmacologia , Selênio/uso terapêutico , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-1beta/metabolismoRESUMO
Protein complexes termed inflammasomes ensure tissue protection from pathogenic and sterile stressors by induction of inflammation. This is mediated by different caspase-1-induced downstream pathways, including activation of the pro-inflammatory cytokines proIL-1ß and -18, induction of a lytic type of cell death, and regulation of the release of other pro-inflammatory molecules. Aberrant inflammasome activation underlies the pathology of numerous (auto)inflammatory diseases. Furthermore, inflammasomes support or suppress tumor development in a complex cell-type- and stage-dependent manner. In human keratinocytes and skin, NLRP1 is the central inflammasome sensor activated by cellular perturbation induced, for example, by UVB radiation. UVB represents the main inducer of skin cancer, which is the most common type of malignancy in humans. Recent evidence demonstrates that activation of NLRP1 in human skin supports the development of cutaneous squamous cell carcinomas (cSCCs) by inducing skin inflammation. In contrast, the NLRP1 inflammasome pathway is restrained in established cSCCs, suggesting that, at this stage, the protein complex has a tumor suppressor role. A better understanding of the complex functions of NLRP1 in the development of cSCCs and in general of inflammasomes in cancer might pave the way for novel strategies for cancer prevention and therapy. These strategies might include stage-specific modulation of inflammasome activation or its downstream pathways by mono- or combination therapy.
Assuntos
Inflamassomos , Neoplasias Cutâneas , Humanos , Inflamassomos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas NLR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caspase 1/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Citocinas/metabolismo , InflamaçãoRESUMO
Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However, aberrant and chronic inflammasome activation underlies the pathology of numerous common inflammatory diseases. Inflammasome assembly causes activation of the protease caspase-1 which in turn activates proinflammatory cytokines and induces a lytic type of cell death termed pyroptosis. Although NLRP1 (NACHT, leucine-rich repeat and pyrin domain containing 1) was the first inflammasome sensor, described almost 20 years ago, the molecular mechanisms underlying its activation and the resulting downstream events are incompletely understood. This is partially a consequence of the poor conservation of the NLRP1 pathway between human and mice. Moreover, recent evidence demonstrates a complex and multi-stage mechanism of NLRP1 inflammasome activation. In contrast to other inflammasome sensors, NLRP1 possesses protease activity required for proteolytic self-cleavage and activation mediated by the function-to-find domain (FIIND). CARD8 is a second FIIND protein and is expressed in humans but not in mice. In immune cells and AML (acute myeloid leukemia) cells, the anti-cancer drug talabostat induces CARD8 activation and causes caspase-1-dependent pyroptosis. In contrast, in human keratinocytes talabostat induces NLRP1 activation and massive proinflammatory cytokine activation. NLRP1 is regarded as the principal inflammasome sensor in human keratinocytes and UVB radiation induces its activation, which is believed to underlie the induction of sunburn. Moreover, gain-of-function mutations of NLRP1 cause inflammatory skin syndromes and a predisposition for the development of skin cancer. SNPs (single nucleotide polymorphisms) of NLRP1 are associated with several (auto)inflammatory diseases with a major skin phenotype, such as psoriasis or vitiligo. Here, we summarize knowledge about NLRP1 with emphasis on its role in human keratinocytes and skin. Due to its accessibility, pharmacological targeting of NLRP1 activation in epidermal keratinocytes represents a promising strategy for the treatment of the numerous patients suffering from NLRP1-dependent inflammatory skin conditions and cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Inflamassomos/metabolismo , Inflamação/patologia , Queratinócitos/patologia , Neoplasias Cutâneas/patologia , Pele/patologia , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Pele/imunologia , Pele/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismoRESUMO
The transcription factor Nrf2 regulates the expression of genes required for protection from xenobiotic and oxidative stress. Under normal conditions Nrf2 is constantly degraded upon ubiquitination, mediated by the Nrf2 inhibitor Keap1. Inflammasomes represent stress-induced protein complexes. They are critically involved in acute and chronic inflammation through caspase-1-mediated activation of pro-inflammatory cytokines. Here, we demonstrate that Nrf2 is a positive regulator of the NLRP3 inflammasome. In contrast, Nrf2-activating compounds, including the anti-inflammatory drug dimethyl fumarate (DMF), inhibit inflammasome activation. Both effects are independent of the transcriptional activity of Nrf2 and, at least in part, not interdependent. On the other hand, NLRP3 inflammasome activation induces a rapid and partly caspase-1- and Keap1-independent degradation of Nrf2. These data argue against a simultaneous activation of both stress-related pathways. Finally, we provide evidence that the cross-regulation of both pathways is controlled by a physical interaction between the Nrf2/Keap1 and NLRP3 complexes.
Assuntos
Inflamassomos/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Caspase 1/metabolismo , Citocinas/imunologia , Fumarato de Dimetilo/farmacologia , Regulação da Expressão Gênica , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Inflamação , Queratinócitos , Camundongos , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The Nrf2 (nuclear factor E2-related factor or nuclear factor (erythroid-derived 2)-like 2) transcription factor is a key player in cytoprotection and activated in stress conditions caused by reactive oxygen species (ROS) or electrophiles. Inflammasomes represent central regulators of inflammation. Upon detection of various stress factors, assembly of the inflamasome protein complex results in activation and secretion of proinflammatory cytokines. In addition, inflammasome activation causes pyroptosis, a lytic form of cell death, which supports inflammation. There is growing evidence of a crosstalk between the Nrf2 and inflammasome pathways at different levels. For example, Nrf2 activating compounds inhibit inflammasomes and consequently inflammation. This review summarizes what is known about the complex and predominantly antagonistic relationship of both stress-activated pathways.
Assuntos
Inflamassomos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Humanos , Inflamassomos/genética , Fator 2 Relacionado a NF-E2/genética , Transdução de SinaisRESUMO
Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and the S100A8 and S100A9 proteins, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1(-/-) mice. IL-18 release was keratinocyte-autonomous, KRT1 and caspase-1 dependent, supporting an upstream role of KRT1 in the pathology. Finally, transcriptome profiling revealed a Krt1-mediated gene expression signature similar to atopic eczema and psoriasis, but different from Krt5 deficiency and epidermolysis bullosa simplex. Our data suggest a functional link between KRT1 and human inflammatory skin diseases.
Assuntos
Inflamação/patologia , Interleucina-18/metabolismo , Queratina-1/metabolismo , Pele/metabolismo , Pele/patologia , Animais , Caspase 1/metabolismo , Diferenciação Celular , Desmossomos/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Inflamação/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Estrutura Quaternária de Proteína , Proteínas S100/metabolismo , Pele/imunologia , Regulação para CimaRESUMO
IL-1ß and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1ß and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1ß activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.
Assuntos
Caspase 1/biossíntese , Caspases Iniciadoras/imunologia , Caspases Iniciadoras/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/genética , Caspase 1/imunologia , Caspase 1/metabolismo , Caspases Iniciadoras/genética , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Humanos , Interleucina-18/biossíntese , Interleucina-18/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Queratinócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pirina , Interferência de RNA , RNA Interferente Pequeno , Raios UltravioletaRESUMO
Interleukin-1α (IL-1α) and -ß both bind to the same IL-1 receptor (IL-1R) and are potent proinflammatory cytokines. Production of proinflammatory (pro)-IL-1α and pro-IL-1ß is induced by Toll-like receptor (TLR)-mediated NF-κB activation. Additional stimulus involving activation of the inflammasome and caspase-1 is required for proteolytic cleavage and secretion of mature IL-1ß. The regulation of IL-1α maturation and secretion, however, remains elusive. IL-1α exists as a cell surface-associated form and as a mature secreted form. Here we show that both forms of IL-1α, the surface and secreted form, are differentially regulated. Surface IL-1α requires NF-κB activation only, whereas secretion of mature IL-1α requires additional activation of the inflammasome and caspase-1. Surprisingly, secretion of IL-1α also required the presence of IL-1ß, as demonstrated in IL-1ß-deficient mice. We further demonstrate that IL-1ß directly binds IL-1α, thus identifying IL-1ß as a shuttle for another proinflammatory cytokine. These results have direct impact on selective treatment modalities of inflammatory diseases.
Assuntos
Inflamação/imunologia , Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Animais , Caspase 1/metabolismo , Citometria de Fluxo , CamundongosRESUMO
Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.
Assuntos
Sistemas CRISPR-Cas , Eletroporação , Edição de Genes , Técnicas de Inativação de Genes , Queratinócitos , RNA Guia de Sistemas CRISPR-Cas , Humanos , Queratinócitos/metabolismo , Queratinócitos/citologia , Eletroporação/métodos , Técnicas de Inativação de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células Cultivadas , Diferenciação Celular/genética , Técnicas de Cultura de Células/métodosRESUMO
Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of CARD8, some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8's different roles in inflammation and inflammatory diseases.
Assuntos
Apoptose , Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Humanos , Inflamassomos/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Animais , Piroptose , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismo , Proteínas de NeoplasiasRESUMO
Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma de Células Escamosas , Humanos , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/patologia , Fibroblastos/patologia , Pele/patologia , DNA Mitocondrial/genética , Microambiente Tumoral/genéticaRESUMO
Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidant defense systems, and activation of this transcription factor is a promising strategy for protection of skin and other organs from environmental insults. To identify efficient Nrf2 activators in keratinocytes, we combined a chemical library screen with computer-based virtual screening. Among 14 novel Nrf2 activators, the most potent compound, a nitrophenyl derivative of 2-chloro-5-nitro-N-phenyl-benzamide, was characterized with regard to its molecular mechanism of action. This compound induced the expression of cytoprotective genes in keratinocytes isolated from wild-type but not from Nrf2-deficient mice. Most importantly, it showed low toxicity and protected primary human keratinocytes from UVB-induced cell death. Therefore, it represents a potential lead compound for the development of drugs for skin protection under stress conditions. Our study demonstrates that chemical library screening combined with advanced computational similarity searching is a powerful strategy for identification of bioactive compounds, and it points toward an innovative therapeutic approach against UVB-induced skin damage.
Assuntos
Benzamidas/farmacologia , Citoproteção/efeitos dos fármacos , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Morte Celular/efeitos da radiação , Linhagem Celular Transformada , Citoproteção/genética , Citoproteção/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Queratinócitos/patologia , Camundongos , Fator 2 Relacionado a NF-E2/genética , Pele/lesões , Pele/metabolismo , Pele/patologiaRESUMO
Gasdermins comprise a family of pore-forming proteins, which play critical roles in (auto)inflammatory diseases and cancer. They are expressed as self-inhibited precursor proteins consisting of an aminoterminal cytotoxic effector domain (NT-GSDM) and a carboxyterminal inhibitor domain (GSDM-CT) separated by an unstructured linker region. Proteolytic processing in the linker region liberates NT-GSDM, which translocates to membranes, forms oligomers, and induces membrane permeabilization, which can disturb the cellular equilibrium that can lead to cell death. Gasdermin activation and pore formation are associated with inflammation, particularly when induced by the inflammatory protease caspase-1 upon inflammasome activation. These gasdermin pores allow the release of the pro-inflammatory cytokines interleukin(IL)-1ß and IL-18 and induce a lytic type of cell death, termed pyroptosis that supports inflammation, immunity, and tissue repair. However, even at the cellular level, the consequences of gasdermin activation are diverse and range from induction of programmed cell death - pyroptosis or apoptosis - to poorly characterized protective mechanisms. The specific effects of gasdermin activation can vary between species, cell types, the membrane that is being permeabilized (plasma membrane, mitochondrial membrane, etc.), and the overall biological state of the local tissue/cells. In epithelia, gasdermins seem to play crucial roles. Keratinocytes represent the main cell type of the epidermis, which is the outermost skin layer with an essential barrier function. Compared to other tissues, keratinocytes express all members of the gasdermin family, in part in a differentiation-specific manner. That raises questions regarding the specific roles of individual GSDM family members in the skin, the mechanisms and consequences of their activation, and the potential crosstalk between them. In this review, we summarize the current knowledge about gasdermins with a focus on keratinocytes and the skin and discuss the possible roles of the different family members in immunity and disease.
RESUMO
Atopic dermatitis is the most common inflammatory skin disease and is characterized by a deficient epidermal barrier and cutaneous inflammation. Genetic studies suggest a key role of keratinocytes in atopic dermatitis pathogenesis, but the alterations in the proteome that occur in the full epidermis have not been defined. Using a pressure-cycling technology and data-independent acquisition approach, we performed quantitative proteomics of epidermis from healthy volunteers and lesional and nonlesional patient skin. Results were validated by targeted proteomics using parallel reaction monitoring mass spectrometry and immunofluorescence staining. Proteins that were differentially abundant in the epidermis of patients with atopic dermatitis versus in healthy control reflect the strong inflammation in lesional skin and the defect in keratinocyte differentiation and epidermal stratification that already characterizes nonlesional skin. Most importantly, they reveal impaired activation of the NRF2-antioxidant pathway and reduced abundance of mitochondrial proteins involved in key metabolic pathways in the affected epidermis. Analysis of primary human keratinocytes with small interfering RNAâmediated NRF2 knockdown revealed that the impaired NRF2 activation and mitochondrial abnormalities are partially interlinked. These results provide insight into the molecular alterations in the epidermis of patients with atopic dermatitis and identify potential targets for pharmaceutical intervention.
Assuntos
Dermatite Atópica , Humanos , Dermatite Atópica/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteômica , Queratinócitos/metabolismo , Epiderme/patologia , Inflamação/patologia , Mitocôndrias/metabolismoRESUMO
Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.