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1.
Int J Biol Sci ; 19(1): 156-166, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36594095

RESUMO

Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.


Assuntos
Carcinoma , Neoplasias Pancreáticas , Neoplasias da Próstata , Masculino , Animais , Camundongos , Humanos , Cromogranina A/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Distribuição Tecidual , Peptídeos/química , Integrinas/metabolismo , Fator de Crescimento Transformador beta/metabolismo
2.
Sci Rep ; 13(1): 282, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609414

RESUMO

Neuroinflammation is a hallmark of hypoxic-ischemic injury and can be characterized by the activation of glial cells and the expression of inflammatory cytokines and chemokines. Interleukin (IL)-1ß and tumor necrosis factor (TNF)α are among the best-characterized early response cytokines and are often expressed concurrently. Several types of central nervous system cells secrete IL-1ß and TNFα, including microglia, astrocytes, and neurons, and these cytokines convey potent pro-inflammatory actions. Chemokines also play a central role in neuroinflammation by controlling inflammatory cell trafficking. Our aim was to characterise the evolution of early neuroinflammation in the neonatal piglet model of hypoxic-ischemic encephalopathy (HIE). Piglets (< 24 h old) were exposed to HI insult, and recovered to 2, 4, 8, 12 or 24H post-insult. Brain tissue from the frontal cortex and basal ganglia was harvested for assessment of glial cell activation profiles and transcription levels of inflammatory markers in HI piglets with comparison to a control group of newborn piglets. Fluorescence microscopy was used to observe microglia, astrocytes, neurons, degenerating neurons and possibly apoptotic cells, and quantitative polymerase chain reaction was used to measure gene expression of several cytokines and chemokines. HI injury was associated with microglial activation and morphological changes to astrocytes at all time points examined. Gene expression analyses of inflammation-related markers revealed significantly higher expression of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin 1 beta (IL-1ß), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)ß in every HI group, with some region-specific differences noted. No significant difference was observed in the level of C-X-C chemokine receptor (CCR)5 over time. This high degree of neuroinflammation was associated with a reduction in the number of neurons in piglets at 12H and 24H in the frontal cortex, and the putamen at 12H. This reduction of neurons was not associated with increased numbers of degenerating neurons or potentially apoptotic cells. HI injury triggered a robust early neuroinflammatory response associated with a reduction in neurons in cortical and subcortical regions in our piglet model of HIE. This neuroinflammatory response may be targeted using novel therapeutics to reduce neuropathology in our piglet model of neonatal HIE.


Assuntos
Citocinas , Hipóxia-Isquemia Encefálica , Animais , Suínos , Citocinas/metabolismo , Animais Recém-Nascidos , Fator de Necrose Tumoral alfa/metabolismo , Doenças Neuroinflamatórias , Neuroglia/metabolismo , Encéfalo/metabolismo , Hipóxia/metabolismo , Microglia/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Fator de Crescimento Transformador beta/metabolismo , Inflamação/patologia
3.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675295

RESUMO

Cardiac fibrosis is a common pathological feature of different cardiovascular diseases, characterized by the aberrant deposition of extracellular matrix (ECM) proteins in the cardiac interstitium, myofibroblast differentiation and increased fibrillar collagen deposition stimulated by transforming growth factor (TGF)-ß activation. Biglycan (BGN), a small leucine-rich proteoglycan (SLRPG) integrated within the ECM, plays a key role in matrix assembly and the phenotypic control of cardiac fibroblasts. Moreover, BGN is critically involved in pathological cardiac remodeling through TGF-ß binding, thus causing myofibroblast differentiation and proliferation. Adenosine receptors (ARs), and in particular A2AR, may play a key role in stimulating fibrotic damage through collagen production/deposition, as a consequence of cyclic AMP (cAMP) and AKT activation. For this reason, A2AR modulation could be a useful tool to manage cardiac fibrosis in order to reduce fibrotic scar deposition in heart tissue. Therefore, the aim of the present study was to investigate the possible crosstalk between A2AR and BGN modulation in an in vitro model of TGF-ß-induced fibrosis. Immortalized human cardiac fibroblasts (IM-HCF) were stimulated with TGF-ß at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. After applying the TGF-ß stimulus, cells were treated with two different A2AR antagonists, Istradefylline and ZM241385, for an additional 24 h, at the concentration of 10 µM and 1 µM, respectively. Both A2AR antagonists were able to regulate the oxidative stress induced by TGF-ß through intracellular reactive oxygen species (ROS) reduction in IM-HCFs. Moreover, collagen1a1, MMPs 3/9, BGN, caspase-1 and IL-1ß gene expression was markedly decreased following A2AR antagonist treatment in TGF-ß-challenged human fibroblasts. The results obtained for collagen1a1, SMAD3, α-SMA and BGN were also confirmed when protein expression was evaluated; phospho-Akt protein levels were also reduced following Istradefylline and ZM241385 use, thus suggesting that collagen production involves AKT recruited by the A2AR. These results suggest that A2AR modulation might be an effective therapeutic option to reduce the fibrotic processes involved in heart pathological remodeling.


Assuntos
Fibroblastos , Proteínas Proto-Oncogênicas c-akt , Humanos , Biglicano/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Colágeno/metabolismo , Fibrose , Adenosina/farmacologia , Adenosina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas
4.
Nat Commun ; 14(1): 349, 2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681697

RESUMO

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-ß activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Humanos , Fator de Crescimento Transformador beta/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/fisiologia , Camadas Germinativas/metabolismo , Mesoderma/metabolismo , Endoderma/metabolismo , Mamíferos/metabolismo
5.
Int J Biol Sci ; 19(1): 204-224, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36594086

RESUMO

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFß1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFß1 leads NDRG1, downstream of GSK3ß, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFß and GSK3ß inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFß to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFß and GSK3ß.


Assuntos
Proteínas de Ciclo Celular , Peptídeos e Proteínas de Sinalização Intracelular , Fator de Crescimento Transformador beta , Neoplasias de Mama Triplo Negativas , Humanos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , NF-kappa B/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
6.
Carbohydr Polym ; 302: 120380, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36604058

RESUMO

Glycans play a crucial role in a variety of physiological and pathological processes. In terms of skin, the sugar chain length, monosaccharide composition and structure of glycans change with age, and thus the changes in glycogens in skin cells are a potential biomarker of aging. The exogenous addition of structurally defined glycans is of great importance for delaying the skin aging process. Fortunately, a functional glycan named manno-oligosaccharide (DOMOS) from Dendrobium officinale was obtained herein by efficient enzymatic depolymerization and exerts anti-aging effects on human skin in vitro and in vivo together with human clinical studies. Further studies show that DOMOS exerts anti-aging effects by triggering the ECM process through a TGF-ß/Smad-SIRT1 signalling pathway. This is the first study to concentrate on the beneficial effects of glycan degradation by a highly specific method on skin aging and provides an all-new solution to the skin aging problem that people are most concerned about.


Assuntos
Transdução de Sinais , Proteínas Smad , Humanos , Proteínas Smad/metabolismo , Sirtuína 1/metabolismo , Sirtuína 1/farmacologia , Envelhecimento , Fator de Crescimento Transformador beta/metabolismo , Polissacarídeos/farmacologia
7.
J Neuroinflammation ; 20(1): 1, 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36593485

RESUMO

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFß levels and membrane expression of TGFß receptors in hippocampus. This leads to microglia activation and reduced Smad7-IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1ß which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFß in the EVs from MSCs, which activates TGFß receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7-IkB pathway. This normalizes IL-1ß, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.


Assuntos
Vesículas Extracelulares , Hiperamonemia , Células-Tronco Mesenquimais , Ratos , Animais , Ratos Wistar , Inflamação/metabolismo , Doenças Neuroinflamatórias , Receptores de N-Metil-D-Aspartato/metabolismo , Hiperamonemia/terapia , Hiperamonemia/metabolismo , NF-kappa B/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Hipocampo/metabolismo , Cognição , Células-Tronco Mesenquimais/metabolismo , Vesículas Extracelulares/metabolismo , Fator de Crescimento Transformador beta/metabolismo
8.
Proc Natl Acad Sci U S A ; 120(3): e2208927120, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36626550

RESUMO

The process of oncogene-induced senescence (OIS) and the conversion between OIS and malignant transformation during carcinogenesis is poorly understood. Here, we show that following overactivation of oncogene Ras in lung epithelial cells, high-level transforming growth factor ß1 (TGF-ß1)-activated SMAD3, but not SMAD2 or SMAD4, plays a determinant role in inducing cellular senescence independent of the p53/p16/p15 senescence pathways. Importantly, SMAD3 binds a potential tumor suppressor ATOH8 to form a transcriptional complex that directly represses a series of cell cycle-promoting genes and consequently causes senescence in lung epithelial cells. Interestingly, the prosenescent SMAD3 converts to being oncogenic and essentially facilitates oncogenic Ras-driven malignant transformation. Furthermore, depleting Atoh8 rapidly accelerates oncogenic Ras-driven lung tumorigenesis, and lung cancers driven by mutant Ras and Atoh8 loss, but not by mutant Ras only, are sensitive to treatment of a specific SMAD3 inhibitor. Moreover, hypermethylation of the ATOH8 gene can be found in approximately 12% of clinical lung cancer cases. Together, our findings demonstrate not only epithelial cellular senescence directed by a potential tumor suppressor-controlled transcriptional program but also an important interplay between the prosenescent and transforming effects of TGF-ß/SMAD3, potentially laying a foundation for developing early detection and anticancer strategies.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Transformação Celular Neoplásica , Genes ras , Proteína Smad3 , Humanos , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Genes Supressores de Tumor , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo
9.
Respir Res ; 24(1): 9, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36627690

RESUMO

We investigated the effects of vegetable glycerin (VG), a main e-cigarette constituent, on endotoxin-induced acute lung injury (ALI). Mice received intratracheal administration of 30% VG in phosphate buffered saline (PBS) vehicle or only PBS (control) for 4 days. On Day 5, mice received an intratracheal instillation of lipopolysaccharide (LPS) (LPS group and VG + LPS group) or PBS (VG group and control group). Lung histopathology, expression of chemokine receptors, and regulatory signaling were analyzed 24 h after the Day 5 treatment. VG significantly increased ALI-associated histopathological and fibrotic changes in both the VG group and LPS-induced ALI mice (VG + LPS group). Immunohistochemistry (IHC) and western blot analyses revealed that VG administration resulted in upregulation of neutrophil markers [lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO)] as well as upregulation of the expression of transforming growth factor-ß (TGF-ß), a central mediator of fibrogenesis, in the lungs of both VG and VG + LPS groups. VG enhanced the expression of adhesion molecules [very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1)] and increased activation of p38 mitogen-activated protein kinase (p38 MAPK) to prompt neutrophil recruitment in the lungs of mice with ALI. Intraperitoneal administration of a p38 inhibitor attenuated these histopathological changes significantly as well as VG-induced upregulation in expression of Ly6G, MPO, VLA-4, VCAM-1, TGF-ß, and collagen-1 in mice with ALI. In conclusion, VG enhances neutrophil chemotaxis and fibrosis and it amplifies the inflammatory response associated with LPS-induced ALI in the lungs via enhancement of p38 MAPK activity.


Assuntos
Lesão Pulmonar Aguda , Sistemas Eletrônicos de Liberação de Nicotina , Glicerol , Animais , Camundongos , Lesão Pulmonar Aguda/metabolismo , Fibrose , Glicerol/efeitos adversos , Integrina alfa4beta1/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Neutrófilos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
10.
Life Sci Alliance ; 6(3)2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36631218

RESUMO

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-ß family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development.


Assuntos
Aminoácidos , Miostatina , Feminino , Gravidez , Animais , Camundongos , Miostatina/genética , Miostatina/química , Aminoácidos/metabolismo , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/química , Músculo Esquelético/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo
11.
Sci Rep ; 13(1): 579, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631456

RESUMO

Iatrogenic injury to endometrial tissue is the main cause of intrauterine adhesions (IUA) and infection can also damage the endometrium. The microbiota plays an important role in the health of the female reproductive tract. However, the mechanism is still unclear. In total, 908 patients with IUA and 11,389 healthy individuals were retrospectively selected for this clinical study. Participant information including vaginal microecological results and human papillomavirus (HPV) status were collected. Univariate and multivariate logistic regression analyses were used to identify the factors related to IUA. Next, animal experiments were performed in a curettage-induced IUA rat model. After the procedure, rats in the experimental group received a vaginal infusion of a Candida albicans (C. albicans) fungal solution. On days 3, 7, and 14 after curettage and infusion, the expression levels of IL-6, fibrotic pathway-related factors (TGF-ß1, Smad 2, and COL1), and estrogen receptor (ER) and progesterone receptor (PR) in rat endometrial tissues were assessed. Fungal infection of the reproductive tract was found to be an independent risk factor for IUA (P < 0.05). The inflammatory response and degree of fibrosis were greater in rats infected with C. albicans than in the controls. The levels of IL-6, TGF-ß1, Smad 2, and COL1 expression in endometrial tissues were significantly higher in the experimental group than in the control group (P < 0.05). However, the ER and PR levels were lower in the IUA group than in the non-IUA group (P < 0.05). C. albicans infection may be related to IUA. C. albicans elicits a strong inflammatory response that can lead to more severe endometrial fibrosis.


Assuntos
Fator de Crescimento Transformador beta1 , Doenças Uterinas , Humanos , Feminino , Ratos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Candida albicans/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Interleucina-6/metabolismo , Regulação para Cima , Estudos Retrospectivos , Doenças Uterinas/patologia , Endométrio/metabolismo , Receptores de Estrogênio/metabolismo , Fibrose , Aderências Teciduais/patologia
12.
Commun Biol ; 6(1): 34, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635368

RESUMO

TGFßs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFß receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFß/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFß1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.


Assuntos
Proteínas Morfogenéticas Ósseas , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas , Ligantes , Proteínas Morfogenéticas Ósseas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta
13.
BMC Biol ; 21(1): 8, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635667

RESUMO

BACKGROUND: Regulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged. RESULTS: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2. CONCLUSIONS: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.


Assuntos
Linfócitos T CD8-Positivos , Linfócitos T Reguladores , Ratos , Animais , Linfócitos T Reguladores/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo
14.
Phytomedicine ; 109: 154590, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36610170

RESUMO

BACKGROUND: Heart failure (HF), caused by stress cardiomyopathy, is a major cause of mortality. Cardiac fibrosis is an essential structural remodeling associated with HF; therefore, preventing cardiac fibrosis is crucial to decelerating the progression of HF. Sodium houttuyfonate (SH), an extract of Houttuynia cordata, has a potent therapeutic effect on hypoxic cardiomyocytes in a myocardial infarction model. PURPOSE: To investigate the preventative and therapeutic effects of SH during isoproterenol (ISO)-induced HF and explore the pharmacological mechanism of SH in alleviating HF. METHODS: We analyzed the overlapping target genes between SH and cardiac fibrosis or HF using a network pharmacology analytical method. We verified the suppressive effect of SH on ISO-induced proliferation and activation of cardiac fibroblasts by immunohistochemical staining and histological analysis in an isoproterenol-induced HF mouse model. Additionally, we investigated the effect of SH by evaluating fibrosis and cardiac remodeling markers. To further decipher the pharmacological mechanism of SH against cardiac fibrosis and HF, we performed a molecular docking analysis between SH and hub common target genes. RESULTS: There were 20 overlapping target genes between SH and cardiac fibrosis and 32 overlapping target genes between SH and HF. The 16 common target genes of SH against cardiac fibrosis and HF included MMP2 (matrix metalloproteinase 2), and p38. SH significantly inhibited the ISO- or TGF-ß-induced expression of Col1α (collagen 1), α-SMA (smooth muscle actin), MMP2, TIMP2 (tissue inhibitor of metalloproteinase 2), TGF-ß (transforming growth factor), and Smad2 phosphorylation. Moreover, both ISO- and TGF-ß-induced p38 phosphorylation was inhibited. Molecular docking analysis showed that SH forms a stable complex with MMP2 and p38. CONCLUSIONS: In addition to protecting cardiomyocytes, SH directly inhibits cardiac fibroblast activation and proliferation by binding to MMP2 and p38, subsequently delaying cardiac fibrosis and HF progression. Our prevention- and intervention-based approaches in this study showed that SH inhibited the development of stress cardiomyopathy-mediated cardiac fibrosis and HF when SH was administered before or after the initiation of cardiac stress.


Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Cardiomiopatia de Takotsubo , Camundongos , Animais , Metaloproteinase 2 da Matriz , Isoproterenol , Inibidor Tecidual de Metaloproteinase-2 , Cardiomiopatia de Takotsubo/patologia , Simulação de Acoplamento Molecular , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fibrose , Fator de Crescimento Transformador beta1/metabolismo , Miocárdio/metabolismo
15.
J Exp Med ; 220(3)2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36598533

RESUMO

Regulatory T cells (Tregs) suppress the activation and subsequent effector functions of CD4 effector T cells (Teffs). However, molecular mechanisms that enforce Treg-mediated suppression in CD4 Teff are unclear. We found that Tregs suppressed activation-induced global protein synthesis in CD4 Teffs prior to cell division. We analyzed genome-wide changes in the transcriptome and translatome of activated CD4 Teffs. We show that mRNAs encoding for the protein synthesis machinery are regulated at the level of translation in activated CD4 Teffs by Tregs. Tregs suppressed global protein synthesis of CD4 Teffs by specifically inhibiting mRNAs of the translation machinery at the level of mTORC1-mediated translation control through concerted action of immunosuppressive cytokines IL-10 and TGFß. Lastly, we found that the therapeutic targeting of protein synthesis with the RNA helicase eIF4A inhibitor rocaglamide A can alleviate inflammatory CD4 Teff activation caused by acute Treg depletion in vivo. These data show that peripheral tolerance is enforced by Tregs through mRNA translational control in CD4 Teffs.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T Reguladores , Ativação Linfocitária , Citocinas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Braz J Med Biol Res ; 55: e12206, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36629522

RESUMO

Although metastasis is the major cause of death in cervical cancer, the mechanism of metastasis is still unclear. The mRNA expression and protein level of latent transforming growth factor beta binding protein 1 (LTBP1) were detected in tumor tissues and paracancerous tissues from in-house samples. Cell proliferation, cell cycle, migration, and in vivo metastasis were determined after LTBP1 was knocked down. Then, 13 drugs were screened, and the changes in cell apoptosis and proliferation and tumor metastasis were detected after drug treatment in shRNA cells. In our in-house samples, LTBP1 was lowly expressed in cervical cancer tissues. After LTBP1 knockdown, cell proliferation was increased, and the ability of in vitro migration and in vivo metastasis was enhanced. At the same time, the proportion of myeloid derived suppressor cells (MDSC) in situ increased, the proportion of T cells decreased, and transforming growth factor beta-1 (TGFß1) signaling was activated. After carboplatin treatment, LTBP1 shRNA cell line apoptosis increased, metastasis in vivo was limited, and the proportion of MDSC in situ decreased. LTBP1 was lowly expressed in cervical cancer, and the inhibition of LTBP1 can improve the malignant degree of the tumor, and this process can be blocked by carboplatin.


Assuntos
Fator de Crescimento Transformador beta , Neoplasias do Colo do Útero , Feminino , Humanos , Fator de Crescimento Transformador beta/metabolismo , Carboplatina , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células
17.
Sci Rep ; 13(1): 25, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36646720

RESUMO

Small airway remodeling (SAR) is a key phenomenon of airflow obstruction in smokers, leading to chronic obstructive pulmonary disease (COPD). SAR results in an increased thickness of small airway walls, with a combination of peribronchiolar fibrosis with increased fibrous tissue and accumulation of mesenchymal and epithelial cells. SAR pathogenesis is still unclear but recent data suggest that alterations in telomerase activity could represent a possible underlying mechanism of SAR. Our study was dedicated to identify a potential protective role of TA-65, a pharmacological telomerase activator, in a cigarette smoke (CS) model of SAR in mice, and to further precise if extra-telomeric effects of telomerase, involving oxidative stress modulation, could explain it. C57BL/6J mice were daily exposed to air or CS during 4 weeks with or without a concomitant administration of TA-65 starting 7 days before CS exposure. Morphological analyses were performed, and mucus production, myofibroblast differentiation, collagen deposition, as well as transforming growth factor-ß1 (TGF-ß1) expression in the small airway walls were examined. In addition, the effects of TA-65 treatment on TGF-ß expression, fibroblast-to-myofibroblast differentiation, reactive oxygen species (ROS) production and catalase expression and activity were evaluated in primary cultures of pulmonary fibroblasts and/or mouse embryonic fibroblasts in vitro. Exposure to CS during 4 weeks induced SAR in mice, characterized by small airway walls thickening and peribronchiolar fibrosis (increased deposition of collagen, expression of α-SMA in small airway walls), without mucus overproduction. Treatment of mice with TA-65 protected them from CS-induced SAR. This effect was associated with the prevention of CS-induced TGF-ß expression in vivo, the blockade of TGF-ß-induced myofibroblast differentiation, and the reduction of TGF-ß-induced ROS production that correlates with an increase of catalase expression and activity. Our findings demonstrate that telomerase is a critical player of SAR, probably through extra-telomeric anti-oxidant effects, and therefore provide new insights in the understanding and treatment of COPD pathogenesis.


Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Telomerase , Camundongos , Animais , Catalase/metabolismo , Telomerase/metabolismo , Remodelação das Vias Aéreas , Fumar Cigarros/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Fibroblastos/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Colágeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fibrose
18.
Cell Mol Life Sci ; 80(2): 43, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36646950

RESUMO

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-ß signaling by directly interacting with TGF-ß type I receptor (TßRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TßRI in the ER. By decreasing cell surface TßRI levels, USP19-ER inhibits TGF-ß/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-ß-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.


Assuntos
Neoplasias da Mama , Fator de Crescimento Transformador beta , Humanos , Feminino , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Transição Epitelial-Mesenquimal/genética , Neoplasias da Mama/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Membrana Celular/metabolismo , Movimento Celular/genética , Linhagem Celular Tumoral , Endopeptidases/metabolismo
19.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36674990

RESUMO

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFß is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvß6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvß6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvß6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvß6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvß6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvß6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.


Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Fator de Crescimento Transformador beta/metabolismo , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Integrinas/metabolismo , Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Microambiente Tumoral
20.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675315

RESUMO

Myofibroblasts escape apoptosis and proliferate abnormally under pathological conditions, especially fibrosis; they synthesize and secrete a large amount of extracellular matrix (ECM), such as α-SMA and collagen, which leads to the distortion of organ parenchyma structure, an imbalance in collagen deposition and degradation, and the replacement of parenchymal cells by fibrous connective tissues. Fibroblast to myofibroblast transition (FMT) is considered to be the main source of myofibroblasts. Therefore, it is crucial to explore the influencing factors regulating the process of FMT for the prevention, treatment, and diagnosis of FMT-related diseases. In recent years, non-coding RNAs, including microRNA, long non-coding RNAs, and circular RNAs, have attracted extensive attention from scientists due to their powerful regulatory functions, and they have been found to play a vital role in regulating FMT. In this review, we summarized ncRNAs which regulate FMT during fibrosis and found that they mainly regulated signaling pathways, including TGF-ß/Smad, MAPK/P38/ERK/JNK, PI3K/AKT, and WNT/ß-catenin. Furthermore, the expression of downstream transcription factors can be promoted or inhibited, indicating that ncRNAs have the potential to be a new therapeutic target for FMT-related diseases.


Assuntos
Miofibroblastos , Fosfatidilinositol 3-Quinases , Humanos , Miofibroblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fibrose
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