RESUMO
TGF-ß type III receptor (TßRIII) is a co-receptor for TGFß family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-ßs, Bone Morphogenetic Proteins (BMP2/4) and Inhibins/Activins regulate different checkpoints during T cell differentiation. We have previously reported that TßRIII modulates T cell development by protecting developing thymocytes from apoptosis, however the role of this co-receptor in peripheral lymphocytes still remains elusive. Here we describe a detailed characterization of TßRIII expression in murine and human lymphocyte subpopulations demonstrating that this co-receptor is significantly expressed in T but not B lymphocytes and among them, preferentially expressed on naïve and central memory T cells. TßRIII was upregulated after TCR stimulation, in parallel to other early activation markers. In contrast, natural and induced Tregs downregulated TßRIII in association with FoxP3 upregulation. Finally, anti-TßRIII blocking experiments demonstrated that TßRIII promotes TGFß-dependent iTreg conversion in vitro, and suggest that this co-receptor may be involved in modulating peripheral T cell tolerance and could be considered as a potential target to boost T cell immune responses.
Assuntos
Proteoglicanas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Memória Imunológica , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Linfócitos T Reguladores/classificação , Linfócitos T Reguladores/metabolismo , Regulação para CimaRESUMO
Galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-ß receptor I (TGF-ßRI), is the only known TGF-ß pathway inhibitor. In the present study, we investigated the effect of galunisertib on taurocholate (TAC)-induced acute pancreatitis (AP) in rats, and the role of TGF-ß and NF-κB signaling pathways. AP was induced by infusion of TAC into the pancreatic duct of Sprague-Dawley male rats (n=30). The rats (220±50 g) were administered galunisertib intragastrically [75 mg·kg-1·day-1 for 2 days (0 and 24 h)]. Serum IL-1ß, IL-6, TNF-α, amylase (AMY), lipase (LIP), and myeloperoxidase (MPO) levels were measured by ELISA. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); NF-κBp65 and TGF-ß1 proteins, as well as TGF-ßRI and p-Smad2/3 proteins, were detected by western blot assay. Cell apoptosis was detected by TUNEL assay. H&E staining was used to evaluate the histopathological alterations of the pancreas. Galunisertib treatment attenuated the severity of AP and decreased the pancreatic histological score. In addition, number of apoptotic cells were significantly increased in the galunisertib-treated group (16.38±2.26) than in the AP group (8.14±1.27) and sham-operated group (1.82±0.73; P<0.05 and P<0.01, respectively). Galunisertib decreased the expression levels of TGF-ßRI and p-Smad2/3 and inhibited NF-κB activation and p65 translocation compared with the sham-operated group. Furthermore, serum IL-1ß, IL-6, TNF-α, AMY and LIP levels and tissue MPO activity were significantly decreased in the galunisertib-treated group. Our data demonstrate that galunisertib attenuates the severity of TAC-induced experimental AP in rats by inducing apoptosis in the pancreas, inhibiting the activation of TGF-ß signals and NF-κB as well as the secretion of pro-inflammatory cytokines.
Assuntos
Pancreatite/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Quinolinas/uso terapêutico , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Doença Aguda , Amilases/sangue , Animais , Apoptose , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Marcação In Situ das Extremidades Cortadas , Interleucina-1beta/sangue , Interleucina-6/sangue , Lipase/sangue , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatite/patologia , Peroxidase/análise , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Reprodutibilidade dos Testes , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangueRESUMO
Studies have showed that there are many biological targets related to the cancer treatment, for example, TGF type I receptor (TGF-ßRI or ALK5). The ALK5 inhibition is a strategy to treat some types of cancer, such as breast, lung, and pancreas. Here, we performed CoMFA and CoMSIA studies for 70 ligands with ALK5 inhibition. The internal validation for both models (q(2)LOO = 0.887 and 0.822, respectively) showed their robustness, while the external validations showed their predictive power (CoMFA: r(2)test = 0.998; CoMSIA: r(2)test = 0.975). After all validations, CoMFA and CoMSIA maps indicated physicochemical evidences on the main factors involved in the interaction between bioactive ligands and ALK5. Therefore, these results suggest molecular modifications to design new ALK5 inhibitors.
Assuntos
Ligantes , Modelos Moleculares , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento Transformadores beta/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reprodutibilidade dos TestesRESUMO
ALK-5 (Activin-Like Kinase 5) is a biological receptor involved in a variety of pathological processes such as cancer and fibrosis. ALK-5 receptor propagates an intracellular signaling that forms a protein complex capable of reaching the nucleus and modulating the gene transcription. In the present study, comparative molecular field analysis (CoMFA) and hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of potent ALK-5 inhibitors. Significant correlation coefficients (CoMFA, r(2)=0.99 and q(2)=0.85; HQSAR, r(2)=0.92 and q(2)=0.72) were obtained, indicating the predictive potential of the 2D and 3D models for untested compounds. The models were then used to predict the potency of a test set, and the predicted values from the HQSAR and CoMFA models were in good agreement with the experimental results. The final QSAR models, along with the information obtained from 3D (steric and electrostatic) contour maps and 2D contribution maps, can be useful for the design of novel bioactive ligands.
Assuntos
Modelos Moleculares , Inibidores de Proteínas Quinases/metabolismo , Ligantes , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidoresRESUMO
OBJECTIVE: Dendritic cells (DCs) modulated with lipopolysaccharide (LPS) are able to reduce inflammation when therapeutically administered into mice with collagen-induced arthritis (CIA). The aim of this study was to uncover the mechanisms that define the tolerogenic effect of short-term LPS-modulated DCs on CIA. METHODS: Bone marrow-derived DCs were stimulated for 4 hours with LPS and characterized for their expression of maturation markers and their cytokine secretion profiles. Stimulated cells were treated with SB203580 or SB431542 to inhibit the p38 or transforming growth factor ß (TGFß) receptor pathway, respectively, or were left unmodified and, on day 35 after CIA induction, were used to inoculate mice. Disease severity was evaluated clinically. CD4+ T cell populations were counted in the spleen and lymph nodes from inoculated or untreated mice with CIA. CD4+ splenic T cells were transferred from mice with CIA treated with LPS-stimulated DCs or from untreated mice with CIA into other mice with CIA on day 35 of arthritis. RESULTS: Treatment with LPS-stimulated DCs increased the numbers of interleukin-10 (IL-10)-secreting and TGFß-secreting CD4+ T cells, but decreased the numbers of Th17 cells. Adoptive transfer of CD4+ T cells from treated mice with CIA reproduced the inhibition of active CIA accomplished with LPS-stimulated DCs. The therapeutic effect of LPS-stimulated DCs and their influence on T cell populations were abolished when the p38 and the TGFß receptor pathways were inhibited. CONCLUSION: DCs modulated short-term (4 hours) with LPS are able to confer a sustained cure in mice with established arthritis by re-educating the CD4+ T cell populations. This effect is dependent on the p38 and the TGFß receptor signaling pathways, which suggests the participation of IL-10 and TGFß in the recovery of tolerance.
Assuntos
Artrite Experimental/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Benzamidas/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Dioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Piridinas/farmacologiaRESUMO
The major neural stem cell population in the developing cerebral cortex is the radial glia cells, which generate neurons and glial cells. The mechanisms that modulate the maintenance of the radial glia stem cell phenotype, or its differentiation, are not completely elucidated. We previously demonstrated that transforming growth factor-ß(1) (TGF-ß(1)) promotes radial glia differentiation into astrocytes in vitro [Glia 2007;55:1023-1033]. Here we investigated the intracellular signaling pathways involved in the TGF-ß(1)-induced radial glia fate commitment. We demonstrate that the mechanisms underlying the TGF-ß(1) effect on radial glia cell differentiation or progenitor potential maintenance diverge. Whereas radial glia differentiation into astrocytes is mediated by the activation of the MAPK signaling pathway, neurogenesis is modulated by different levels of PI3K and SMAD2/3 activity. Our work demonstrates that radial glia cells are a heterogeneous population and a potential target of TGF-ß(1), and suggests that its effect on radial glia fate commitment is mediated by the recruitment of a complex multipathway mechanism that controls astrocyte and neuronal generation in the developing cerebral cortex.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Neuroglia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Análise de Variância , Animais , Astrócitos/citologia , Benzamidas/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Dioxóis/farmacologia , Flavonoides/farmacologia , Imuno-Histoquímica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-â (TGF-â). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-â T. cruzi, or SB-431542, an inhibitor of TGF-â receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-â signalling had been shown previously to be highly activated. We demonstrated that TGF-â treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-â secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-â levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.
Assuntos
Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Benzamidas/uso terapêutico , Doença de Chagas/metabolismo , /metabolismo , Dioxóis/uso terapêutico , Junções Comunicantes/metabolismo , Miócitos Cardíacos/química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/uso terapêutico , Doença de Chagas/tratamento farmacológico , Imunofluorescência , Junções Comunicantes/efeitos dos fármacos , Imuno-Histoquímica , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
Betaglycan is a coreceptor for members of the transforming growth factor beta (TGF-beta) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Delta10 and Sol Delta11, bind TGF-beta, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-beta activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K(d)'s in the low nanomolar range for the three TGF-beta isoforms, while those for Sol Delta10 and Sol Delta11 are 1-2 orders of magnitude higher. SPR analysis further shows that the K(d)'s of Sol Delta11 are not changed in the presence of Sol Delta10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-beta through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-beta neutralizing activity.
Assuntos
Testes de Neutralização , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Fibrinolisina/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/químicaRESUMO
Chagas' disease induced by Trypanosoma cruzi infection is an important cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. We previously reported that transforming growth factor beta (TGF-beta) is implicated in several regulatory aspects of T. cruzi invasion and growth and in host tissue fibrosis. This prompted us to evaluate the therapeutic action of an inhibitor of TGF-beta signaling (SB-431542) administered during the acute phase of experimental Chagas' disease. Male Swiss mice were infected intraperitoneally with 10(4) trypomastigotes of T. cruzi (Y strain) and evaluated clinically for the following 30 days. SB-431542 treatment significantly reduced mortality and decreased parasitemia. Electrocardiography showed that SB-431542 treatment was effective in protecting the cardiac conduction system. By 14 day postinfection, enzymatic biomarkers of tissue damage indicated that muscle injury was decreased by SB-431542 treatment, with significantly lower blood levels of aspartate aminotransferase and creatine kinase. In conclusion, inhibition of TGF-beta signaling in vivo appears to potently decrease T. cruzi infection and to prevent heart damage in a preclinical mouse model. This suggests that this class of molecules may represent a new therapeutic agent for acute and chronic Chagas' disease that warrants further clinical exploration.
Assuntos
Benzamidas/uso terapêutico , Cardiomiopatia Chagásica/prevenção & controle , Doença de Chagas/tratamento farmacológico , Dioxóis/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Bradicardia/prevenção & controle , Masculino , Camundongos , Miocárdio/patologia , Parasitemia/tratamento farmacológico , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/fisiologiaRESUMO
Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-beta (TGF-beta). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-beta T. cruzi, or SB-431542, an inhibitor of TGF-beta receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-beta signalling had been shown previously to be highly activated. We demonstrated that TGF-beta treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-beta secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-beta levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.
Assuntos
Benzamidas/uso terapêutico , Doença de Chagas/metabolismo , Conexina 43/metabolismo , Dioxóis/uso terapêutico , Junções Comunicantes/metabolismo , Miócitos Cardíacos/química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/uso terapêutico , Adulto , Animais , Doença de Chagas/tratamento farmacológico , Feminino , Imunofluorescência , Junções Comunicantes/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Confocal , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
The antiinflammatory cytokine transforming growth factor beta (TGF-beta) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-beta type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-beta pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-beta signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-beta signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.