RESUMEN
Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-ß (TGF-ß), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-ß is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-ß signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-ß neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 104 parasites from the Y strain and C57BL/6 mice infected with 102 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-ß signaling pathways in T. cruzi-infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß neutralization.
Asunto(s)
Cardiomiopatía Chagásica , Enfermedad de Chagas , Trypanosoma cruzi , Ratones , Animales , Factor de Crecimiento Transformador beta/metabolismo , Cardiomiopatía Chagásica/tratamiento farmacológico , Trypanosoma cruzi/metabolismo , Ratones Endogámicos C57BL , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , FibrosisRESUMEN
Transforming growth factor beta (TGF-ß) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-ß; (ii) the potential involvement of TGF-ß pathway on T. cruzi invasion of host cells; (iii) association of TGF-ß with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-ß to treat the cardiac alterations of Chagas disease-affected patients.
Asunto(s)
Cardiomiopatía Chagásica , Trypanosoma cruzi , Cardiomiopatía Chagásica/tratamiento farmacológico , Cardiomiopatía Chagásica/metabolismo , Corazón , Humanos , Miocardio/patología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Trypanosoma cruzi/fisiologíaRESUMEN
Transforming growth factor beta (TGF-β) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-β; (ii) the potential involvement of TGF-β pathway on T. cruzi invasion of host cells; (iii) association of TGF-β with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-β to treat the cardiac alterations of Chagas disease-affected patients.
RESUMEN
The anti-inflammatory cytokine transforming growth factor beta (TGF-ß) plays an important role in Chagas disease (CD), a potentially life-threatening illness caused by Trypanosoma cruzi. In this review we revisited clinical studies in CD patients combined with in vitro and in vivo experiments, presenting three main sections: an overview of epidemiological, economic, and clinical aspects of CD and the need for new biomarkers and treatment; a brief panorama of TGF-ß roles and its intracellular signaling pathways, and an update of what is known about TGF-ß and Chagas disease. In in vitro assays, TGF-ß increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-ß modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-ß signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-ß1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-ß as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-ß pathway improved several cardiac electric parameters, reversed the loss of connexin-43 enriched intercellular plaques, reduced fibrosis of the cardiac tissue, restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Finally, TGF-ß polymorphisms indicate that CD immunogenetics is at the base of this phenomenon. We searched in a Brazilian population five single-nucleotide polymorphisms (-800 G>A rs1800468, -509 C>T rs1800469, +10 T>C rs1800470, +25 G>C rs1800471, and +263 C>T rs1800472), showing that CD patients frequently express the TGF-ß1 gene genotypes CT and TT at position -509, as compared to noninfected persons; similar results were observed with genotypes TC and CC at codon +10 of the TGF-ß1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-ß1 polymorphisms are associated with Chagas disease susceptibility. Studies in genetically different populations susceptible to CD will help to gather new insights and encourage the use of TGF-ß as a CD biomarker.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Animales , Biomarcadores , Enfermedad de Chagas/parasitología , Humanos , Inmunogenética , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Trypanosoma cruzi/metabolismoRESUMEN
Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000-8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.
Asunto(s)
Telangiectasia Hemorrágica Hereditaria/tratamiento farmacológico , Telangiectasia Hemorrágica Hereditaria/metabolismo , Bevacizumab/uso terapéutico , Reposicionamiento de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína Smad4/metabolismo , Tacrolimus , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/metabolismoRESUMEN
The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for Bmp9. We found that Bmp9 deletion led to premature mortality only in the 129/Ola strain. We have previously shown that Bmp9 deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus Bmp9-KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.
Asunto(s)
Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Cirrosis Hepática , Hígado/metabolismo , Animales , Biomarcadores/metabolismo , Capilares/metabolismo , Capilares/patología , Diferenciación Celular , Células Endoteliales/patología , Factor 2 de Diferenciación de Crecimiento/genética , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.
Asunto(s)
Benzamidas/uso terapéutico , Cardiomiopatía Chagásica/tratamiento farmacológico , Corazón/efectos de los fármacos , Pirazoles/uso terapéutico , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Tripanocidas/uso terapéutico , Animales , Cardiomiopatía Chagásica/parasitología , Cardiomiopatía Chagásica/patología , Enfermedad Crónica , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrosis/tratamiento farmacológico , Corazón/parasitología , Sistema de Conducción Cardíaco/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Carga de Parásitos , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.
Asunto(s)
Receptores de Activinas Tipo II/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Factor 2 de Diferenciación de Crecimiento/genética , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/citología , Factor 2 de Diferenciación de Crecimiento/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteómica , ARN Mensajero/genética , Distribución Aleatoria , Estadísticas no Paramétricas , Técnicas de Cultivo de Tejidos/métodosRESUMEN
Altered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Estabilidad del ARN , Factores Asociados con la Proteína de Unión a TATA/fisiología , Animales , Células COS , Carcinogénesis/metabolismo , Células Cultivadas , Chlorocebus aethiops , Femenino , Mutación con Ganancia de Función/fisiología , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estabilidad del ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Factores Asociados con la Proteína de Unión a TATA/genética , Dedos de Zinc/genéticaRESUMEN
We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFß canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFß1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.
Asunto(s)
Neoplasias Encefálicas/genética , Antígeno CD47/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteína smad3/genética , Trombospondina 1/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Antígeno CD47/antagonistas & inhibidores , Antígeno CD47/metabolismo , Línea Celular Tumoral , Corteza Cerebral , Glioblastoma/irrigación sanguínea , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Captura por Microdisección con Láser , Masculino , Ratones , Ratones Noqueados , Invasividad Neoplásica , Péptidos/farmacología , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína smad3/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Análisis de Supervivencia , Trombospondina 1/antagonistas & inhibidores , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RATIONALE: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. OBJECTIVE: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. METHOD AND RESULTS: The absence of BMP9 in Bmp9-/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9-/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates. CONCLUSIONS: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.
Asunto(s)
Factor 2 de Diferenciación de Crecimiento/antagonistas & inhibidores , Hipertensión Pulmonar/prevención & control , Receptores de Activinas Tipo II/farmacología , Animales , Células Cultivadas , Endotelina-1/genética , Factor 2 de Diferenciación de Crecimiento/fisiología , Humanos , Hipoxia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas WistarRESUMEN
Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.
Asunto(s)
Diferenciación Celular , Células Endoteliales/citología , Factor 2 de Diferenciación de Crecimiento/farmacología , Linfangiogénesis , Células Madre Embrionarias de Ratones/citología , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Calcineurina/metabolismo , Proliferación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Vasos Linfáticos/citología , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Endotelio Vascular/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Multimerización de Proteína , Células 3T3 , Animales , Proteínas Morfogenéticas Óseas/sangre , Proteínas Morfogenéticas Óseas/química , Endotelio Vascular/citología , Factor 2 de Diferenciación de Crecimiento/sangre , Factor 2 de Diferenciación de Crecimiento/química , Factores de Diferenciación de Crecimiento/sangre , Factores de Diferenciación de Crecimiento/química , Humanos , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
Transforming growth factor ß1 (TGF-ß1) is an important mediator in Chagas disease. Furthermore, patients with higher TGF-ß1 serum levels show a worse clinical outcome. Gene polymorphism may account for differences in cytokine production during infectious diseases. We tested whether TGFB1 polymorphisms could be associated with Chagas disease susceptibility and severity in a Brazilian population. We investigated five single-nucleotide polymorphisms (-800 G>A, -509 C>T, +10 T>C, +25 G>C, and +263 C>T). 152 patients with Chagas disease (53 with the indeterminate form and 99 with the cardiac form) and 48 noninfected subjects were included. Genotypes CT and TT at position -509 of the TGFB1 gene were more frequent in Chagas disease patients than in noninfected subjects. Genotypes TC and CC at codon +10 of the TGFB1 gene were also more frequent in Chagas disease patients than in noninfected subjects. We found no significant differences in the distribution of the studied TGFB1 polymorphisms between patients with the indeterminate or cardiac form of Chagas disease. Therefore, -509 C>T and +10 T>C TGFB1 polymorphisms are associated with Chagas disease susceptibility in a Brazilian population.
Asunto(s)
Enfermedad de Chagas/genética , Polimorfismo de Nucleótido Simple , Factor de Crecimiento Transformador beta/genética , Adulto , Anciano , Brasil , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR-483-5p and miR-139-5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA-mRNA pairs in ACC. We identified N-myc downstream-regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR-139-5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR-483-5p and miR-139-5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR-139-5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR-483-5p or miR-139-5p and their targets was demonstrated in reporter assays. Downregulation of miR-483-5p or miR-139-5p in the ACC cell lines NCI-H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage-independent growth. MiR-483-5p or miR-139-5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial-to-mesenchymal transition are downstream effectors of miR-483-5p/NDRG2 and miR-139-5p/NDRG4 pathways. Collectively, our data show for the first time that miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Regulación Neoplásica de la Expresión Génica , Genes myc , MicroARNs/fisiología , Familia de Multigenes , Regiones no Traducidas 3' , Apoptosis/fisiología , Adhesión Celular/fisiología , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Células HEK293 , Humanos , MicroARNs/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Estadificación de Neoplasias , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Pronóstico , ARN Mensajero/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Preeclampsia is a gestational pathology that complicates 2 to 8% of pregnancies and is one of the major causes of maternal and fetal morbidity and mortality worldwide. The aim of this work was to study the epidemiological profile of preeclampsia in Casablanca and to identify risk factors as well as factors of poor maternal and fetal prognosis. 401 preeclamptic cases were collected in the gynecology-obstetrics "C" Service of Lalla Meryem Maternity of Ibn Rochd University Hospital of Casablanca (2010-2011) were included in this study and a statistical analysis with the SPSS software version (16.0) was performed. We used the Chi-2 test to analyze qualitative variables and Student's test and ANOVA (analysis of variance) for quantitative variables. The incidence of preeclampsia was (7.1%). The epidemiological profile was that of a primipara (57.6%), average age 30 years and (66.8%) of pregnancies were not followed. Multiparity was a factor of poor maternal prognosis (p = 0.007). The low gestational age and no prenatal care were factors of maternal as well as fetal prognosis. Risk factors frequently found in our patients were obesity (15.21%) and chronic hypertension (5.73%) as vascular-renal history; abortion (16.46%) and perinatal death (5.24%) as obstetric history. Preeclampsia is a common obstetric pathology in our context. Better prenatal care and early diagnosis could reduce its incidence.
Asunto(s)
Edad Gestacional , Preeclampsia/epidemiología , Atención Prenatal/métodos , Adolescente , Adulto , Femenino , Hospitales Universitarios , Humanos , Hipertensión/epidemiología , Incidencia , Persona de Mediana Edad , Marruecos/epidemiología , Obesidad/epidemiología , Preeclampsia/diagnóstico , Preeclampsia/etiología , Embarazo , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3'-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.
RESUMEN
We describe the design and optimization of polyfunctional scaffolds based on a fluorescent indolizine core derivatized with various orthogonal groups (amines, esters, oximes, alkynes, etc.). To show one application as tools in biology, the scaffold was used to prepare drug-biotin conjugates that were then immobilized onto avidin-agarose for affinity chromatography. More specifically, the antiangiogenic drug COB223, whose mechanism of action remained unclear, was chosen as a proof-of-concept drug. The drug-selective discrimination of proteins observed after elution of the cell lysates through the affinity columns, functionalized either with the biologically active COB223 or a structurally related inactive analogue (COB236), is a clear indication that the presence of the indolizine core does not limit drug-protein interaction and confirms the usefulness of the indolizine scaffold. Furthermore, the separation of COB223-interacting proteins from human placental extracts unveiled unanticipated protein targets belonging to the family of regulatory RNA-binding proteins, which opens the way to new hypotheses on the mode of action of this antiangiogenic drug.
RESUMEN
TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.