RESUMO
OBJECTIVE: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (ß-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (ß-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , beta-Arrestina 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , RNA/metabolismoRESUMO
RATIONALE: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. OBJECTIVE: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. METHODS AND RESULTS: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. CONCLUSIONS: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.
Assuntos
Indutores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Imidazóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , PPAR gama/metabolismo , Piperazinas/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Artéria Pulmonar/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo , PPAR gama/genética , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated ß1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.
Assuntos
Retrovirus Endógenos , Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Antivirais , Elafina/genética , Elafina/metabolismo , Elafina/farmacologia , Retrovirus Endógenos/metabolismo , Hipertensão Pulmonar Primária Familiar/genética , Humanos , Hipertensão Pulmonar/genética , Integrinas/genética , Integrinas/metabolismo , Elastase de Leucócito/metabolismo , Camundongos , Neutrófilos/metabolismo , Proteômica , Vinculina/genética , Vinculina/metabolismoRESUMO
Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.
Assuntos
Pneumonia , Lesão Pulmonar Induzida por Ventilação Mecânica , Humanos , Camundongos , Animais , Receptores de Esfingosina-1-Fosfato , Caderinas , Esfingosina/farmacologia , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lisofosfolipídeos/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Proteína ADAM10 , Proteínas de Membrana , Secretases da Proteína Precursora do AmiloideRESUMO
BACKGROUND: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. METHODS: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. RESULTS: ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was ß-catenin dependent. CONCLUSIONS: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.
Assuntos
Aldeído Oxirredutases/genética , Regulação da Expressão Gênica , Hipertensão Arterial Pulmonar/genética , Aldeído Oxirredutases/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Músculo Liso/patologia , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.
Assuntos
Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Hipoglicemiantes/farmacologia , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Líquidos Corporais/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/química , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Modelos Moleculares , Obesidade/induzido quimicamente , Obesidade/metabolismo , Osteogênese/efeitos dos fármacos , PPAR gama/agonistas , PPAR gama/química , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Rosiglitazona , Tiazolidinedionas/efeitos adversos , Tiazolidinedionas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Aumento de Peso/efeitos dos fármacosRESUMO
Mitochondria are highly dynamic organelles essential for cell metabolism, growth, and function. It is becoming increasingly clear that endothelial cell dysfunction significantly contributes to the pathogenesis and vascular remodeling of various lung diseases, including pulmonary arterial hypertension (PAH), and that mitochondria are at the center of this dysfunction. The more we uncover the role mitochondria play in pulmonary vascular disease, the more apparent it becomes that multiple pathways are involved. To achieve effective treatments, we must understand how these pathways are dysregulated to be able to intervene therapeutically. We know that nitric oxide signaling, glucose metabolism, fatty acid oxidation, and the TCA cycle are abnormal in PAH, along with alterations in the mitochondrial membrane potential, proliferation, and apoptosis. However, these pathways are incompletely characterized in PAH, especially in endothelial cells, highlighting the urgent need for further research. This review summarizes what is currently known about how mitochondrial metabolism facilitates a metabolic shift in endothelial cells that induces vascular remodeling during PAH.
Assuntos
Hipertensão Pulmonar , Doenças Vasculares , Humanos , Hipertensão Pulmonar/metabolismo , Remodelação Vascular , Células Endoteliais/metabolismo , Pulmão/metabolismo , Estresse Oxidativo , Doenças Vasculares/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Proliferação de CélulasRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Camundongos , Humanos , Animais , Hipertensão Arterial Pulmonar/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Artéria Pulmonar/metabolismo , Dano ao DNA , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7ß), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.
Assuntos
Neoplasias , Pequeno RNA não Traduzido , Animais , Cromatina , Humanos , Mamíferos/genética , Neoplasias/genética , Isoformas de Proteínas/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismoRESUMO
We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response-88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.
Assuntos
Retrovirus Endógenos , Transição Epitelial-Mesenquimal/imunologia , Hipertensão Pulmonar , Macrófagos/imunologia , Monócitos/imunologia , Artéria Pulmonar , Pirofosfatases/metabolismo , Animais , Antígeno CD146/metabolismo , Retrovirus Endógenos/metabolismo , Retrovirus Endógenos/patogenicidade , Células Endoteliais/metabolismo , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/virologia , Inflamação/metabolismo , Inflamação/virologia , Camundongos , Artéria Pulmonar/imunologia , Artéria Pulmonar/patologia , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismoRESUMO
The human metabolome is the cumulative product of ingested metabolites and those produced by the body and its microbiota. Together these metabolites can dynamically report on the health and disease state of an individual, as well as their response to drug treatments and other external perturbations. Profiling metabolites in human body fluids provides an opportunity to identify biomarkers and stratify patients for personalized treatments but requires the development of high-throughput approaches compatible with large cohort and longitudinal studies. Here we review in detail sample preparation and analytical liquid chromatography-mass spectrometry (LC-MS) methods to measure the broad chemical diversity of metabolites found in human plasma and urine.
Assuntos
Cromatografia Líquida , Metabolômica/métodos , Medicina de Precisão/métodos , Espectrometria de Massas em Tandem , Biomarcadores/sangue , Líquidos Corporais/metabolismo , Fezes/química , Humanos , Marcação por Isótopo , Metaboloma/genéticaRESUMO
Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinson's disease and schizophrenia.
Assuntos
Simulação de Acoplamento Molecular , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/química , Sítios de Ligação , Ácidos Graxos Insaturados/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Ligação ProteicaRESUMO
Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.
Assuntos
Reparo do DNA , Células Endoteliais/metabolismo , Homeostase , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Instabilidade Genômica , Células HEK293 , Humanos , Modelos Biológicos , Ligação Proteica , Artéria Pulmonar/patologia , Transdução de Sinais , UbiquitinaçãoRESUMO
The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.
Assuntos
Proteína Oncogênica tpr-met/química , Proteína Oncogênica tpr-met/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Humanos , Zíper de Leucina , Modelos Moleculares , Mutação , Proteína Oncogênica tpr-met/genética , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estabilidade ProteicaRESUMO
Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.
Assuntos
DNA/metabolismo , Complexos Multiproteicos/química , Coativador 2 de Receptor Nuclear/química , Coativador 2 de Receptor Nuclear/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Medição da Troca de Deutério , Regulação da Expressão Gênica , Humanos , Ligantes , PPAR gama/química , PPAR gama/metabolismo , Ligação Proteica , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismoRESUMO
Blocking phosphorylation of peroxisome proliferator-activated receptor (PPAR)γ at Ser(273) is one of the key mechanisms for antidiabetes drugs to target PPARγ. Using high-throughput phosphorylation screening, we here describe that Gleevec blocks cyclin-dependent kinase 5-mediated PPARγ phosphorylation devoid of classical agonism as a PPARγ antagonist ligand. In high fat-fed mice, Gleevec improved insulin sensitivity without causing severe side effects associated with other PPARγ-targeting drugs. Furthermore, Gleevec reduces lipogenic and gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues. Interestingly, Gleevec increases browning of white adipose tissue and energy expenditure. Taken together, the results indicate that Gleevec exhibits greater beneficial effects on both glucose/lipid metabolism and energy homeostasis by blocking PPARγ phosphorylation. These data illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type 2 diabetes.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Resistência à Insulina , PPAR gama/antagonistas & inibidores , Células 3T3-L1 , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/fisiologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPARγ), to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.
RESUMO
A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses.
Assuntos
Receptor X Retinoide alfa/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Clonagem Molecular , Cristalografia por Raios X , Regulação da Expressão Gênica/fisiologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , PPAR gama/genética , PPAR gama/metabolismo , Conformação Proteica , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptor X Retinoide alfa/genéticaRESUMO
The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.
RESUMO
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently, we reported the development of PPARγ antagonist SR1664, designed to block the obesity-induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin-sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow-derived mesenchymal stem cells with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand-mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.