RESUMO
Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , Sequência de Bases , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Epiteliais/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Metotrexato/farmacologia , Mutação/genética , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Piroptose/efeitos dos fármacos , Piroptose/genética , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Diapausa , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Células Clonais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Irinotecano/farmacologia , Irinotecano/uso terapêutico , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
Assuntos
Cérebro/patologia , Proteína Semelhante a ELAV 4/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Neurônios/patologia , Organoides/metabolismo , Splicing de RNA/genética , Proteínas tau/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Biomarcadores/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Hidrazonas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Grânulos de Estresse/efeitos dos fármacos , Grânulos de Estresse/metabolismo , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.
Assuntos
Encéfalo/metabolismo , Neurocinina B/metabolismo , Precursores de Proteínas/metabolismo , Isolamento Social , Estresse Psicológico , Taquicininas/metabolismo , Animais , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/patologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurocinina B/genética , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Taquicininas/antagonistas & inibidores , Receptores de Taquicininas/metabolismo , Taquicininas/antagonistas & inibidores , Taquicininas/genética , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. METHODS: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. RESULTS: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. CONCLUSIONS: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.
Assuntos
Vesículas Extracelulares , Insuficiência Cardíaca , Quinolonas , Animais , Masculino , Camundongos , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Vesículas Extracelulares/efeitos dos fármacos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/prevenção & controle , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Distribuição Aleatória , Regulação para Cima/efeitos dos fármacos , MicroRNAs , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismoRESUMO
BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
Assuntos
Antineoplásicos , Desmetilação , Síndromes Mielodisplásicas , Oncogenes , Regulação para Cima , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desmetilação/efeitos dos fármacos , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oncogenes/efeitos dos fármacos , Oncogenes/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.
Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Oxirredutases Intramoleculares/metabolismo , Leucócitos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Pericitos/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Anticorpos Bloqueadores/farmacologia , Arteríolas/imunologia , Capilares/imunologia , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Leucócitos/efeitos dos fármacos , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vênulas/imunologiaRESUMO
Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.
Assuntos
Diferenciação Celular , Flavonoides , Metiltransferases , Osteogênese , Ratos Sprague-Dawley , Animais , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ratos , Feminino , Flavonoides/farmacologia , Metiltransferases/metabolismo , Metiltransferases/genética , Humanos , Osteoporose/patologia , Osteoporose/metabolismo , Osteoporose/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Ovariectomia , Regulação para Cima/efeitos dos fármacos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Adenosina/análogos & derivados , Adenosina/metabolismoRESUMO
The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.
Assuntos
Transição Epitelial-Mesenquimal , Ácido Láctico , Lipopolissacarídeos , Transportadores de Ácidos Monocarboxílicos , Fibrose Pulmonar , Simportadores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Simportadores/metabolismo , Simportadores/genética , Simportadores/antagonistas & inibidores , Camundongos , Ácido Láctico/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/induzido quimicamente , Camundongos Endogâmicos C57BL , Linhagem Celular , Masculino , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.
Assuntos
Compostos Benzidrílicos , Glucosídeos , Hipertensão , Ratos Endogâmicos SHR , Ratos Wistar , Inibidores do Transportador 2 de Sódio-Glicose , Trocador 3 de Sódio-Hidrogênio , Regulação para Cima , Animais , Masculino , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Glucosídeos/farmacologia , Compostos Benzidrílicos/farmacologia , Regulação para Cima/efeitos dos fármacos , Ratos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Rim/efeitos dos fármacosRESUMO
Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.
Assuntos
Desoxicitidina , Gencitabina , Proteômica , Proteínas Quinases Associadas a Fase S , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Proteômica/métodos , Linhagem Celular Tumoral , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Regulação para Cima/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Studies have shown that adoptive transfer of myeloid-derived suppressor cells (MDSCs) can alleviate various inflammatory diseases, including glomerulonephritis, but the long-term effects of the transferred MDSCs are still unclear. In addition, although glucocorticoids exert immunosuppressive effects on inflammatory diseases by inducing the expansion of MDSCs, the impact of glucocorticoids on the immunosuppressive function of MDSCs and their molecular mechanisms are unclear. In this study, we found that adoptive transfer of MDSCs to doxorubicin-induced focal segmental glomerulosclerosis (FSGS) mice for eight consecutive weeks led to an increase in serum creatinine and proteinuria and aggravation of renal interstitial fibrosis. Similarly, 8 weeks of high-dose dexamethasone administration exacerbated renal interstitial injury and interstitial fibrosis in doxorubicin-induced mice, manifested as an increase in serum creatinine and proteinuria, collagen deposition and α-SMA expression. On this basis, we found that dexamethasone could enhance MDSC expression and secretion of the fibrosis-related cytokines TGF-ß and IL-10. Mechanistically, we revealed that dexamethasone promotes the expression of immunoglobulin-like transcription factor 4 (ILT4), which enhances the T-cell inhibitory function of MDSCs and promotes the activation of STAT6, thereby strengthening the expression and secretion of TGF-ß and IL-10. Knocking down ILT4 alleviated renal fibrosis caused by adoptive transfer of MDSCs. Therefore, our findings demonstrate that the role and mechanism of dexamethasone mediate the expression and secretion of TGF-ß and IL-10 in MDSCs by promoting the expression of ILT4, thereby leading to renal fibrosis.
Assuntos
Dexametasona , Fibrose , Células Supressoras Mieloides , Animais , Dexametasona/farmacologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/efeitos dos fármacos , Camundongos , Rim/patologia , Rim/metabolismo , Rim/efeitos dos fármacos , Masculino , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Camundongos Endogâmicos C57BL , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Transferência Adotiva , Modelos Animais de Doenças , Regulação para Cima/efeitos dos fármacos , Interleucina-10/metabolismo , Interleucina-10/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.
Assuntos
Apoptose , Proteólise , Proteína Supressora de Tumor p53 , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Animais , Camundongos , Linhagem Celular Tumoral , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/efeitos dos fármacos , Ligação ProteicaRESUMO
Mitoxantrone (MX) is an effective treatment for breast cancer; however, high efflux of MX that is accomplished by breast cancer resistance protein (BCRP) leads to acquired multidrug resistance (MDR), reducing MX's therapeutic efficacy in breast cancer. Non-muscle myosin IIA (NMIIA) and its heavy phosphorylation at S1943 have been revealed to play key roles in tumor metastasis and progression, including in breast cancer; however, their molecular function in BCRP-mediated MDR in breast cancer remains unknown. In this study, we revealed that the expression of NMIIA heavy chain phosphorylation at S1943 was downregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and stable expression of NMIIA-S1943A mutant increased BCRP expression and promoted the resistance of MCF-7/MX cells to MX. Meanwhile, NMIIA S1943 phosphorylation induced by epidermal growth factor (EGF) was accompanied by the downregulation of BCRP in MCF-7/MX cells. Furthermore, stable expression of NMIIA-S1943A in MCF-7/MX cells resulted in upregulation of N-cadherin and the accumulation of ß-catenin on the cell surface, which inhibited the nucleus translocation of ß-catenin and Wnt/ß-catenin-based proliferative signaling. EGF stimulation of MCF-7/MX cells showed the downregulation of N-cadherin and ß-catenin. Our results suggest that decreased NMIIA heavy phosphorylation at S1943 increases BCRP expression and promotes MX resistance in breast cancer cells via upregulating N-cadherin expression.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Neoplasias da Mama , Caderinas , Resistencia a Medicamentos Antineoplásicos , Mitoxantrona , Proteínas de Neoplasias , Regulação para Cima , Humanos , Mitoxantrona/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Fosforilação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação para Cima/efeitos dos fármacos , Caderinas/metabolismo , Caderinas/genética , Células MCF-7 , Antineoplásicos/farmacologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Ferroptosis is a pattern of cell death caused by iron-dependent accumulation of lipid peroxides and is closely associated with the occurrence and development of multiple diseases. Acrolein (ACR), one of the final metabolites of lipid peroxidation, is a reactive carbonyl species with strong biotoxicity. Effective detection of ACR is important for understanding its role in the progression of ferroptosis and studying the specific mechanisms of ferroptosis-mediated diseases. However, visualization detection of ACR during ferroptosis has not yet been reported. In this work, the first ratiometric fluorescent probe (HBT-SH) based on 2-(2'-hydroxyphenyl) benzothiazole (HBT) was designed for tracing endogenous ACR with an unprecedented regiospecific ACR-induced intramolecular cyclization strategy, which employs 2-aminoethanethiol as an ACR-selective recognition receptor. The experimental results showed that HBT-SH has excellent selectivity, high sensitivity (LOD = 0.26 µM) and good biocompatibility. More importantly, the upregulation of ACR levels was observed during ferroptosis in HeLa cells and zebrafish, indicating that ACR may be a specific active molecule that plays an essential biological role during ferroptosis or may serve as a potential marker of ferroptosis, which has great significance for studying the pathological process and treatment options of ferroptosis-related diseases.
Assuntos
Acroleína , Ferroptose , Corantes Fluorescentes , Peixe-Zebra , Ferroptose/efeitos dos fármacos , Acroleína/química , Acroleína/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Células HeLa , Animais , Regulação para Cima/efeitos dos fármacos , Imagem Óptica , Estrutura MolecularRESUMO
Endoplasmic reticulum stress (ERS) plays a crucial role in the pathogenesis of diabetic nephropathy (DN), and it is often accompanied by an increase in reactive oxygen species (ROS) production. However, the precise relationship between NFE2-related factor-2 (Nrf2), a key regulator of ROS balance, and ERS in DN remains elusive. This study aimed to investigate the impact of Nrf2 on ERS and its therapeutic potential in DN. Herein, ERS-related changes, including increased activating transcription factor-6 (ATF6), glucose-regulated protein 78 (GRP78), and transcription factor C/EBP homologous protein (CHOP) expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose cultured human renal proximal tubular (HK-2) cells. Nrf2 knockdown increased the sensitivity of HK-2 cells to ERS under high glucose conditions, underscoring the regulatory role of Nrf2 in ERS modulation. Notably, upregulating Nrf2 in ezetimibe-treated diabetic mice restored ERS markers and ameliorated albuminuria, glomerular hypertrophy, mesangial expansion, and tubulointerstitial fibrosis. Furthermore, the inhibition of ERS in HK-2 cells by the ROS scavenger, N-acetylcysteine, highlights the interplay between ROS and ERS. This study, for the first time, elucidates that the upregulation of Nrf2 may alleviate the negative influence of ROS-mediated ERS, presenting a promising therapeutic avenue for delaying the progression of DN. These findings suggest a potential strategy for targeting Nrf2 and ERS in developing novel therapeutic interventions for DN.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Espécies Reativas de Oxigênio , Regulação para Cima , Animais , Humanos , Masculino , Camundongos , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/tratamento farmacológico , Chaperona BiP do Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Polycystic ovary syndrome (PCOS), a prevalent endocrine disorder among women of reproductive age, is characterized by disturbances in hormone levels and ovarian dysfunction. Ferroptosis, a unique form of regulated cell death characterized by iron-dependent lipid peroxidation. Emerging evidence indicates that ferroptosis may have a significant role in the pathogenesis of PCOS, highlighting the importance of studying this mechanism to better understand the disorder and potentially develop novel therapeutic interventions. METHODS: To create an in vivo PCOS model, mice were injected with dehydroepiandrosterone (DHEA) and the success of the model was confirmed through further assessments. Ferroptosis levels were evaluated through detecting ferroptosis-related indicators. Ferroptosis-related genes were found through bioinformatic analysis and identified by experiments. An in vitro PCOS model was also established using DHEA treated KGN cells. The molecular binding relationship was confirmed using a chromatin immunoprecipitation (ChIP) assay. RESULTS: In PCOS model, various ferroptosis-related indicators such as MDA, Fe2+, and lipid ROS showed an increase, while GSH, GPX4, and TFR1 exhibited a decrease. These findings indicate an elevated level of ferroptosis in the PCOS model. The ferroptosis-related gene FADS2 was identified and validated. FADS2 and PPAR-α were shown to be highly expressed in ovarian tissue and primary granulosa cells (GCs) of PCOS mice. Furthermore, the overexpression of both FADS2 and PPAR-α in KGN cells effectively suppressed the DHEA-induced increase in ferroptosis-related indicators (MDA, Fe2+, and lipid ROS) and the decrease in GSH, GPX4, and TFR1 levels. The ferroptosis agonist erastin reversed the suppressive effect, suggesting the involvement of ferroptosis in this process. Additionally, the FADS2 inhibitor SC26196 was found to inhibit the effect of PPAR-α on ferroptosis. Moreover, the binding of PPAR-α to the FADS2 promoter region was predicted and confirmed. This indicates the regulatory relationship between PPAR-α and FADS2 in the context of ferroptosis. CONCLUSIONS: Our study indicates that PPAR-α may have an inhibitory effect on DHEA-induced ferroptosis in GCs by enhancing the expression of FADS2. This discovery provides valuable insights into the pathophysiology and potential therapeutic targets for PCOS.
Assuntos
Ácidos Graxos Dessaturases , Ferroptose , Células da Granulosa , PPAR alfa , Síndrome do Ovário Policístico , Regulação para Cima , Animais , Feminino , Camundongos , Desidroepiandrosterona/farmacologia , Modelos Animais de Doenças , Ferroptose/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Síndrome do Ovário Policístico/genética , PPAR alfa/metabolismo , PPAR alfa/genética , Regulação para Cima/efeitos dos fármacos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismoRESUMO
Artificial sweeteners, which contain no or few calories, have been widely used in various foods and beverages, and are regarded as safe alternatives to sugar by the Food and Drug Administration. While several studies suggest that artificial sweeteners are not related to cancer development, some research has reported their potential association with the risk of cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether acesulfame potassium (Ace K), a commonly used artificial sweetener, induces immune evasion of HCC cells by upregulating programmed death ligand-1 (PD-L1). Ace K elevated the protein levels of PD-L1 in HCC cells without increasing its mRNA levels. The upregulation of PD-L1 protein levels in HCC cells by Ace K was induced by attenuated autophagic degradation of PD-L1, which was mediated by the Ace K-stimulated ERK1/2-mTORC1 signaling pathway. Ace K-induced upregulation of PD-L1 attenuated T cell-mediated death of HCC cells, thereby promoting immune evasion of HCC cells. In summary, the present study suggests that Ace K promotes HCC progression by upregulating the PD-L1 protein level.
Assuntos
Autofagia , Antígeno B7-H1 , Carcinoma Hepatocelular , Neoplasias Hepáticas , Tiazinas , Regulação para Cima , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Autofagia/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Tiazinas/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Linhagem Celular Tumoral , Edulcorantes/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with ß-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.
Assuntos
Ecdisterona/farmacologia , Proteínas de Insetos/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Clatrina/metabolismo , Ecdisterona/química , Ecdisterona/metabolismo , Endocitose , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Cerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood-brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs. METHODS: Using the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNß or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro. RESULTS: In vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNß, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNß or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo. CONCLUSION: Our study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.