RESUMEN

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2 to 3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over one year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR-ISR axis could be a potential therapeutic strategy for PVOD.

RESUMEN

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving vascular remodeling in PVOD. Here we show that administration of MMC in rats mediates activation of protein kinase R (PKR) and the integrated stress response (ISR), which leads to the release of the endothelial adhesion molecule vascular endothelial (VE) cadherin (VE-Cad) in complex with RAD51 to the circulation, disruption of endothelial barrier and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates VE-Cad depletion, elevation of vascular permeability and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of the receptor BMPR2, underscoring the role of deregulated bone morphogenetic protein signaling in the development of PVOD.

Asunto(s)

Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Modelos Animales de Enfermedad , Fenotipo , Enfermedad Veno-Oclusiva Pulmonar , Animales , Enfermedad Veno-Oclusiva Pulmonar/genética , Enfermedad Veno-Oclusiva Pulmonar/tratamiento farmacológico , Enfermedad Veno-Oclusiva Pulmonar/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Remodelación Vascular/efectos de los fármacos , Cadherinas/genética , Cadherinas/metabolismo , Humanos , Masculino , Antígenos CD/metabolismo , Antígenos CD/genética , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Mutación , Permeabilidad Capilar/efectos de los fármacos , Ratas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico

RESUMEN

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.

RESUMEN

Drosha is a core component of the Microprocessor complex that cleaves primary-microRNAs (pri-miRNAs) to generate precursor-miRNA and regulates the expression of ∼80 ribosomal protein (RP) genes. Despite the fact that mutations in the amino-terminal region of Drosha (Drosha-NTR) are associated with a vascular disorder, hereditary hemorrhagic telangiectasia, the precise function of Drosha-NTR remains unclear. By deleting exon 5 from the Drosha gene and generating a Drosha mutant lacking the NTR (ΔN), we demonstrate that ΔN is unable to process pri-miRNAs, which leads to a global miRNA depletion, except for the miR-183/96/182 cluster. We find that Argonaute 2 facilitates the processing of the pri-miR-183/96/182 in ΔN cells. Unlike full-length Drosha, ΔN is not degraded under serum starvation, resulting in unregulated RP biogenesis and protein synthesis in ΔN cells, allowing them to evade growth arrest. This study reveals the essential role of Drosha-NTR in miRNA production and nutrient-dependent translational control.

RESUMEN

SMAD4 mutations that disrupt its interaction with SMAD3 and attenuate tumor suppression by TGF-ß are major oncogenic drivers. Tang et al. (2020) report the discovery of small molecules that restore the SMAD4:SMAD3 complex and its cytostatic activity, exemplifying the therapeutic potential of fixing tumor suppressor mutants using molecular glues.

Asunto(s)

Transducción de Señal , Transactivadores , Genes Supresores de Tumor , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/genética

RESUMEN

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

Asunto(s)

Biosíntesis de Proteínas , Proteínas Ribosómicas/biosíntesis , Ribosomas , Animales , Eritropoyesis , Ratones , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo

RESUMEN

PURPOSE OF REVIEW: The TGFß (transforming growth factor ß) superfamily - a large group of structurally related and evolutionarily conserved proteins - profoundly shapes and organizes the vasculature during normal development and adult homeostasis. Mutations inactivating several of its ligands, receptors, or signal transducers set off hereditary hemorrhagic telangiectasia (HHT), a disorder that causes capillary networks to form incorrectly. Drosha, an essential microRNA-processing enzyme, also interfaces with TGFß signal transducers, but its involvement in vascular conditions had not been tested until recently. This review summarizes current evidence that links mutations of Drosha to HHT. RECENT FINDINGS: Genetic studies have revealed that rare missense mutations in the Drosha gene occur more commonly among HHT patients than in healthy people. Molecular analyses also indicated that Drosha enzymes with HHT-associated mutations generate microRNAs less efficiently than their wild-type counterpart when stimulated by TGFß ligands. In zebrafish or mouse, mutant Drosha proteins cause the formation of dilated, leaky blood vessels deprived of capillaries, similar to those typically found in patients with HHT. SUMMARY: Recent evidence suggests that Drosha-mediated microRNA biogenesis contributes significantly to the control of vascular development and homeostasis by TGFß. Loss or reduction of Drosha function may predispose carriers to HHT and possibly other vascular diseases.

Asunto(s)

MicroARNs , Mutación Missense , Ribonucleasa III , Transducción de Señal , Telangiectasia Hemorrágica Hereditaria , Animales , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Telangiectasia Hemorrágica Hereditaria/enzimología , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/patología , Pez Cebra

RESUMEN

The integrity of blood vessels is fundamental to vascular homeostasis. Inactivating mutations in the bone morphogenetic protein (BMP) receptor type II (BMPR2) gene cause hereditary vascular disorders, including pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia, suggesting that BMPR2 and its downstream signaling pathway are pivotal to the maintenance of vascular integrity through an unknown molecular mechanism. Here we report that inactivation of BMPR2 in pulmonary vascular endothelial cells results in a deficit of RAD51, an enzyme essential for DNA repair and replication. Loss of RAD51, which causes DNA damage and cell death, is also detected in animal models and human patients with pulmonary arterial hypertension. Restoration of BMPR2 or activation of the BMP signaling pathway rescues RAD51 and prevents DNA damage. This is an unexpected role of BMP signaling in preventing the accumulation of DNA damage and the concomitant loss of endothelial integrity and vascular remodeling associated with vascular disorders.

RESUMEN

The transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-ß or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-ß and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an overrepresentation of rare nonsynonymous variants of DROSHA Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino-directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.

Asunto(s)

Regulación de la Expresión Génica , Mutación , Neovascularización Patológica , Ribonucleasa III/genética , Ribonucleasa III/fisiología , Telangiectasia Hemorrágica Hereditaria/genética , Animales , Estudios de Casos y Controles , Células Cultivadas , Niño , Estudios de Cohortes , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Morfogénesis , Linaje , Fenotipo , Ribonucleasa III/metabolismo , Transducción de Señal , Telangiectasia Hemorrágica Hereditaria/metabolismo , Telangiectasia Hemorrágica Hereditaria/patología , Pez Cebra/embriología , Pez Cebra/fisiología

RESUMEN

Hematopoietic stem and progenitor cells arise from the vascular endothelium of the dorsal aorta and subsequently switch niche to the fetal liver through unknown mechanisms. Here we report that vascular endothelium-specific deletion of mouse Drosha (Drosha cKO), an enzyme essential for microRNA biogenesis, leads to anemia and death. A similar number of hematopoietic stem and progenitor cells emerge from Drosha-deficient and control vascular endothelium, but Drosha cKO-derived hematopoietic stem and progenitor cells accumulate in the dorsal aorta and fail to colonize the fetal liver. Depletion of the let-7 family of microRNAs is a primary cause of this defect, as it leads to activation of leukotriene B4 signaling and induction of the α4ß1 integrin cell adhesion complex in hematopoietic stem and progenitor cells. Inhibition of leukotriene B4 or integrin rescues maturation and migration of Drosha cKO hematopoietic stem and progenitor cells to the fetal liver, while it hampers hematopoiesis in wild-type animals. Our study uncovers a previously undefined role of innate leukotriene B4 signaling as a gatekeeper of the hematopoietic niche transition.Hematopoietic stem and progenitor cells are generated first from the vascular endothelium of the dorsal aorta and then the fetal liver but what regulates this switch is unknown. Here, the authors show that changing miRNA biogenesis and leukotriene B4 signaling in mice modulates this switch in the niche.

Asunto(s)

Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Leucotrieno B4/metabolismo , MicroARNs/genética , Nicho de Células Madre/genética , Animales , Aorta/metabolismo , Endotelio Vascular/metabolismo , Hígado/embriología , Hígado/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Transducción de Señal/genética

RESUMEN

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics.

Asunto(s)

Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/fisiopatología , Locomoción/fisiología , Sinapsis/patología , Algoritmos , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/fisiología , Conducta Animal/efectos de los fármacos , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Drosophila/efectos de los fármacos , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Ensayos Analíticos de Alto Rendimiento , Larva/efectos de los fármacos , Larva/fisiología , Quinasas Lim/antagonistas & inhibidores , Quinasas Lim/genética , Quinasas Lim/metabolismo , Masculino , Ratones , Ratones Noqueados , Bibliotecas de Moléculas Pequeñas/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo

RESUMEN

Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morphogenetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin reorganization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism.

Asunto(s)

Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Síndrome del Cromosoma X Frágil/genética , Animales , Trastorno Autístico/genética , Encéfalo/metabolismo , Cofilina 1/metabolismo , Cruzamientos Genéticos , Drosophila melanogaster , Síndrome del Cromosoma X Frágil/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Heterocigoto , Humanos , Quinasas Lim/metabolismo , Ratones , Ratones Noqueados , Neuritas/metabolismo , Neuronas/metabolismo , Fosforilación , Plásmidos/metabolismo , Corteza Prefrontal/metabolismo , Dominios Proteicos , ARN Interferente Pequeño/metabolismo , Transducción de Señal

RESUMEN

It is widely accepted that different forms of stress activate a common target, p53, yet different outcomes are triggered in a stress-specific manner. For example, activation of p53 by genotoxic agents, such as camptothecin (CPT), triggers apoptosis, while non-genotoxic activation of p53 by Nutlin-3 (Nut3) leads to cell-cycle arrest without significant apoptosis. Such stimulus-specific responses are attributed to differential transcriptional activation of various promoters by p53. In this study, we demonstrate that CPT, but not Nut3, induces miR-203, which downregulates anti-apoptotic bcl-w and promotes cell death in a p53-dependent manner. We find that acetylation of K120 in the DNA-binding domain of p53 augments its association with the Drosha microprocessor and promotes nuclear primary miRNA processing. Knockdown of human orthologue of Males absent On the First (hMOF), the acetyltransferase that targets K120 in p53, abolishes induction of miR-203 and cell death mediated by CPT. Thus, this study reveals that p53 acetylation at K120 plays a critical role in the regulation of the Drosha microprocessor and that post-transcriptional regulation of gene expression by p53 via miRNAs plays a role in determining stress-specific cellular outcomes.

Asunto(s)

Daño del ADN , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Camptotecina/farmacología , Supervivencia Celular/genética , Células HCT116/efectos de los fármacos , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Imidazoles/metabolismo , MicroARNs/genética , Piperazinas/metabolismo , Regiones Promotoras Genéticas , Procesamiento Postranscripcional del ARN , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Proteína p53 Supresora de Tumor/genética

RESUMEN

The signaling pathway mediated by BMPs plays an essential role during development as well as the maintenance of homeostasis in adult. Aberrant activation or inactivation of BMP signaling can lead to developmental defects and various human disorders. To fine-tune its activity, BMP signaling is regulated both positively and negatively by extrinsic and intrinsic regulatory factors that modulate binding of ligand to the receptors, and the activity of receptors and their dedicated signal transducers, the Smad proteins. Upon BMP binding to the receptor complex, Smad proteins translocate to the nucleus and modulate gene expression transcriptionally by directly associating with the promoter region of target genes, or post-transcriptionally through modulation of microRNA (miRNA) synthesis. In this study, we demonstrate that BMP signaling down-regulates transcription of the miRNA-302∼367 gene cluster. We show that the type II BMP receptor (BMPRII) is a novel target of miR-302. Upon overexpression, miR-302 targets a partially complementary sequence localized in the 3'-untranslated region (UTR) of BMPRII transcripts and leads to destabilization of the transcripts and down-regulation of BMP signaling. We propose that the negative regulatory loop of BMP4-miR-302-BMPRII is a potential mechanism for the maintenance and fine-tuning of the BMP signaling pathway in various systems.

Asunto(s)

Proteína Morfogenética Ósea 4/metabolismo , MicroARNs/antagonistas & inhibidores , Regiones no Traducidas 3' , Animales , Aorta/citología , Células COS , Chlorocebus aethiops , Homeostasis , Humanos , Ratones , Ratones Endogámicos C3H , MicroARNs/química , MicroARNs/metabolismo , Músculo Liso/metabolismo , Miocitos del Músculo Liso/citología , Interferencia de ARN , Transducción de Señal , Transcripción Genética

RESUMEN

Pulmonary artery hypertension (PAH) patients exhibit elevated levels of inflammatory cytokines and infiltration of inflammatory cells in the lung. Concurrently, mutations of bmpr2, the gene encoding the type II receptor of bone morphogenetic proteins (BMP), are found in ∼75% of patients with familial PAH, but a possible nexus between increased inflammation and diminished BMP signaling has hitherto remained elusive. We previously showed that BMP4 triggers nuclear localization of the Myocardin-related transcription factor A (MRTF-A) in human pulmonary artery smooth muscle cells (PASMC), resulting in the induction of contractile proteins. Here we report the BMPR2-dependent repression of a set of inflammatory mediators in response to BMP4 stimulation of PASMC. Forced expression of MRTF-A precisely emulates the anti-inflammatory effect of BMP4, while MRTF-A depletion precludes BMP4-mediated cytokine inhibition. BMP4 and MRTF-A block signaling through NF-κB, the keystone of most pathways leading to inflammatory responses, at the level of chromatin recruitment and promoter activation. Moreover, MRTF-A physically interacts with RelA/p65, the NF-κB subunit endowed with a transcription activation domain. Interestingly, the MRTF-A-NF-κB interaction is mutually antagonistic: stimulation of NF-κB signaling by TNFα, as well as p65 overexpression, hinders MRTF-A activity and the expression of contractile genes. Thus, a molecular inhibitory pathway linking BMP4 signaling, activation of MRTF-A, and inhibition of NF-κB provides insights into the etiology of PAH and a potential focus of therapeutic intervention.

Asunto(s)

Proteína Morfogenética Ósea 4/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipertensión Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Transducción de Señal , Proteína Morfogenética Ósea 4/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/terapia , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Proteínas de Fusión Oncogénica/genética , Transactivadores , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética

RESUMEN

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.

Asunto(s)

Proteína Morfogenética Ósea 4/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , MicroARNs/metabolismo , Contracción Muscular/fisiología , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Morfogenética Ósea 4/genética , Movimiento Celular/fisiología , Proteínas Activadoras de GTPasa/genética , Humanos , MicroARNs/genética , Proteínas Musculares/genética , Músculo Liso Vascular/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo

RESUMEN

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.

Asunto(s)

Proteínas Argonautas/metabolismo , MicroARNs/metabolismo , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Animales , Proteínas Argonautas/genética , Hipoxia de la Célula/genética , Células Cultivadas , Gránulos Citoplasmáticos/metabolismo , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Hidroxilación , Pulmón/citología , Pulmón/metabolismo , Masculino , MicroARNs/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Cultivo Primario de Células , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Transporte de Proteínas , Arteria Pulmonar/citología , Ratas , Ribonucleasa III/metabolismo

RESUMEN

In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a "contractile" phenotype are required for the normal regulation of vascular tone. The transforming growth factor-ß (TGF-ß) superfamily of growth factors (TGF-ßs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-ß or BMP has not been explored. Here, we demonstrate that TGF-ß and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-ß or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-ß and BMP4. Interestingly, both TGF-ß and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-ß mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-ß and BMP4 signaling in the regulation of KLF4 and contractile genes.

Asunto(s)

Proteína Morfogenética Ósea 4/metabolismo , Núcleo Celular/metabolismo , Regulación hacia Abajo/fisiología , Factores de Transcripción de Tipo Kruppel/biosíntesis , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteína Morfogenética Ósea 4/genética , Línea Celular , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , MicroARNs/genética , Contracción Muscular/fisiología , Proteínas Musculares/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética/fisiología

RESUMEN

Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ∼60% of familial PAH (FPAH) and ∼30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calcium-calcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH.

Asunto(s)

Amilorida/análogos & derivados , Proteínas Morfogenéticas Óseas/metabolismo , Pulmón/irrigación sanguínea , Factores de Transcripción NFATC/metabolismo , Arteria Pulmonar/fisiología , Transducción de Señal/efectos de los fármacos , Canales Iónicos Sensibles al Ácido , Amilorida/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Canales de Sodio/metabolismo , Activación Transcripcional/efectos de los fármacos

RESUMEN

The signal transducers of the transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP), the Smads, promote the expression of a subset of miRNAs by facilitating the cleavage reaction by Drosha. The mechanism that limits Smad-mediated processing to a selective group of miRNAs remained hitherto unexplored. In this study, we expand the number of TGFbeta/BMP-regulated miRNAs (T/B-miRs) to 20. Of interest, a majority of T/B-miRs contain a consensus sequence (R-SBE) within the stem region of the primary transcripts of T/B-miRs (pri-T/B-miRs). Here, we demonstrate that Smads directly bind the R-SBE. Mutation of the R-SBE abrogates TGFbeta/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs and impairs their processing, whereas introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and to allow regulation by TGFbeta/BMP. Thus, Smads are multifunctional proteins that modulate gene expression transcriptionally through DNA binding and posttranscriptionally through pri-miRNA binding and regulation of miRNA processing.

Asunto(s)

MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Ribonucleasa III/metabolismo , Proteínas Smad/metabolismo , Células Cultivadas , Humanos , Metaloproteinasas de la Matriz Secretadas/genética , Metaloproteinasas de la Matriz Secretadas/metabolismo , MicroARNs/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Proteínas/genética , Proteínas/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas de Unión al ARN , Ribonucleasa III/genética , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/farmacología