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1.
Front Endocrinol (Lausanne) ; 14: 1166076, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37388215

RESUMO

Introduction: Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Methods: Following the identification of a loss-of-function variant (p.Arg703Gln) in the peptidylglycine a-amidating monooxygenase (PAM) gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated PA kindreds for PAM variants. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. Results: In germline DNA, we detected seven heterozygous, likely pathogenic missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with growth hormone excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, splicing by minigene assays, and amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs with diagnoses linked to pituitary gland hyperfunction. Conclusion: The identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.


Assuntos
Doenças da Hipófise , Neoplasias Hipofisárias , Criança , Humanos , Variações do Número de Cópias de DNA , Hipófise , Neoplasias Hipofisárias/genética , Oxigenases de Função Mista
2.
medRxiv ; 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36711613

RESUMO

Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

3.
Circ Res ; 131(1): 77-90, 2022 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-35534923

RESUMO

BACKGROUND: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. METHODS: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. RESULTS: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Col1a2, Fn1, etc) and tissue inhibitor of metalloproteinase 3 (Timp3) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. CONCLUSIONS: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.


Assuntos
Aterosclerose , MicroRNAs , Placa Aterosclerótica , Antagomirs/metabolismo , Antagomirs/uso terapêutico , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/terapia , Humanos , Macrófagos/metabolismo , MicroRNAs/metabolismo , Placa Aterosclerótica/patologia
4.
Nat Commun ; 13(1): 107, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013237

RESUMO

Aging is impacted by interventions across species, often converging on metabolic pathways. Transcription factors regulate longevity yet approaches for their pharmacological modulation to exert geroprotection remain sparse. We show that increased expression of the transcription factor Grainyhead 1 (GRH-1) promotes lifespan and pathogen resistance in Caenorhabditis elegans. A compound screen identifies FDA-approved drugs able to activate human GRHL1 and promote nematodal GRH-1-dependent longevity. GRHL1 activity is regulated by post-translational lysine methylation and the phosphoinositide (PI) 3-kinase C2A. Consistently, nematodal longevity following impairment of the PI 3-kinase or insulin/IGF-1 receptor requires grh-1. In BXD mice, Grhl1 expression is positively correlated with lifespan and insulin sensitivity. In humans, GRHL1 expression positively correlates with insulin receptor signaling and also with lifespan. Fasting blood glucose levels, including in individuals with type 2 diabetes, are negatively correlated with GRHL1 expression. Thereby, GRH-1/GRHL1 is identified as a pharmacologically malleable transcription factor impacting insulin signaling and lifespan.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Classe II de Fosfatidilinositol 3-Quinases/genética , Diabetes Mellitus Tipo 2/genética , Fator de Crescimento Insulin-Like I/genética , Insulina/metabolismo , Longevidade/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Glicemia/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Humanos , Resistência à Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade/efeitos dos fármacos , Metilação , Camundongos , Papaverina/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Vorinostat/farmacologia
5.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34782454

RESUMO

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3ß-hydroxysterol Δ24-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45+CD11b+ cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.


Assuntos
Aterosclerose/tratamento farmacológico , Desmosterol/farmacologia , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Ativação de Macrófagos/efeitos dos fármacos , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Colesterol/metabolismo , Vasos Coronários , Células Espumosas/metabolismo , Humanos , Inflamação/metabolismo , Metabolismo dos Lipídeos , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Placa Aterosclerótica/metabolismo , Esteróis/metabolismo
6.
EMBO Mol Med ; 9(9): 1244-1262, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28674080

RESUMO

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR-21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR-21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR-21 expression influences foam cell formation, sensitivity to ER-stress-induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR-21 in macrophages increases the expression of the miR-21 target gene, MKK3, promoting the induction of p38-CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post-translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR-21 in atherogenesis.


Assuntos
Apoptose , Aterosclerose/fisiopatologia , Macrófagos/imunologia , MicroRNAs/imunologia , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Vasos Sanguíneos/imunologia , Feminino , Humanos , MAP Quinase Quinase 3/genética , MAP Quinase Quinase 3/imunologia , Macrófagos/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Necrose/genética , Necrose/imunologia , Necrose/patologia , Necrose/fisiopatologia
7.
Cell Rep ; 19(13): 2743-2755, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28658622

RESUMO

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14α-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages in vitro, decreases IFNß-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNß-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.


Assuntos
Lanosterol/imunologia , Macrófagos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Regulação para Baixo , Feminino , Técnicas de Inativação de Genes , Humanos , Imunidade Inata/efeitos dos fármacos , Lanosterol/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esterol 14-Desmetilase/imunologia
8.
Nat Commun ; 7: 12313, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27460411

RESUMO

Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.


Assuntos
Proteína 4 Semelhante a Angiopoietina/deficiência , Aterosclerose/metabolismo , Aterosclerose/patologia , Progressão da Doença , Células-Tronco Hematopoéticas/metabolismo , Monócitos/metabolismo , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Apoptose , Aterosclerose/complicações , Transplante de Medula Óssea , Proliferação de Células , Sobrevivência Celular , Células Espumosas/metabolismo , Humanos , Inflamação/complicações , Inflamação/patologia , Leucocitose/complicações , Leucocitose/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células Progenitoras Mieloides/metabolismo , Placa Aterosclerótica/complicações , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia
9.
Circ Res ; 118(1): 38-47, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26472816

RESUMO

RATIONALE: Several lines of evidence indicate that the regulation of microRNA (miRNA) levels by different stimuli may contribute to the modulation of stimulus-induced responses. The miR-17-92 cluster has been linked to tumor development and angiogenesis, but its role in vascular endothelial growth factor-induced endothelial cell (EC) functions is unclear and its regulation is unknown. OBJECTIVE: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17-92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17-92 cluster in the endothelium (miR-17-92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. METHODS AND RESULTS: Here, we show that Erk/Elk1 activation on VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (chromatin immunoprecipitation analysis) of the miR-17-92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17-92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Finally, we provide genetic evidence that miR-17-92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN (phosphatase and tensin homolog) is a target of the miR-17-92 cluster and is a crucial mediator of miR-17-92-induced EC proliferation. However, the angiogenic transcriptional program is reduced when miR-17-92 is inhibited. CONCLUSIONS: Taken together, our results indicate that VEGF-induced miR-17-92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis.


Assuntos
Endotélio Vascular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , MicroRNAs/biossíntese , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neovascularização Fisiológica/efeitos dos fármacos
10.
Atherosclerosis ; 243(2): 499-509, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26520906

RESUMO

RATIONALE: Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. METHODS AND RESULTS: Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50-fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10-20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. CONCLUSIONS: The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/sangue , Dieta Hiperlipídica , Fígado/metabolismo , MicroRNAs/metabolismo , Receptores de LDL/metabolismo , Regiões 3' não Traduzidas , Transportador 1 de Cassete de Ligação de ATP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores/sangue , Células COS , Chlorocebus aethiops , Biologia Computacional , Bases de Dados Genéticas , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Hep G2 , Humanos , Macaca mulatta , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Receptores de LDL/genética , Fatores de Tempo , Transfecção , Triglicerídeos/sangue
11.
Nat Med ; 21(11): 1280-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26437365

RESUMO

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , MicroRNAs/genética , Receptores de LDL/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Regulação da Expressão Gênica , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Receptores de LDL/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
12.
Am J Pathol ; 185(11): 3090-101, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26348575

RESUMO

The hypoxia-inducible factor (Hif)-1α (Hif-1α) and Hif-2α (Epas1) have a critical role in both normal development and cancer. von Hippel Lindau (Vhl) protein, encoded by a tumor suppressor gene, is an E3 ubiquitin ligase that targets Hif-1α and Epas1 to the proteasome for degradation. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Human tumors of the soft tissue are a very complex and heterogeneous group of neoplasias. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fibrose/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais , Neoplasias de Tecidos Moles/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fibrose/metabolismo , Fibrose/prevenção & controle , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/prevenção & controle , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
13.
FASEB J ; 29(2): 597-610, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25392271

RESUMO

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2(-/-) mice to Ldlr(-/-) mice results in marked reduction of the progression of atherosclerosis compared with Ldlr(-/-) mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2(-/-) macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.


Assuntos
Aterosclerose/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Glicemia/metabolismo , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Movimento Celular , Colesterol/metabolismo , Citocinas/metabolismo , Progressão da Doença , Inflamação , Insulina/química , Leucócitos/citologia , Lipídeos/sangue , Lipoproteínas LDL/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Placa Aterosclerótica , Receptores de LDL/genética
14.
Dev Biol ; 393(1): 124-36, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24972088

RESUMO

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.


Assuntos
Células-Tronco Mesenquimais/citologia , Osteogênese/genética , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Condrogênese/genética , Condrogênese/fisiologia , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/crescimento & desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Botões de Extremidades/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Supressora de Tumor Von Hippel-Lindau/genética
15.
J Cell Mol Med ; 18(6): 1104-12, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24636235

RESUMO

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155(-/-) ) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155(-/-) mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155(-/-) mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.


Assuntos
Derme/metabolismo , Derme/patologia , MicroRNAs/fisiologia , Cicatrização/genética , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Derme/lesões , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Cell Sci ; 127(Pt 6): 1169-78, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24463821

RESUMO

MicroRNA-149 (miR-149) is located within the first intron of the glypican-1 (GPC1) gene. GPC1 is a low affinity receptor for fibroblast growth factor (FGF2) that enhances FGF2 binding to its receptor (FGFR1), subsequently promoting FGF2-FGFR1 activation and signaling. Using bioinformatic approaches, both GPC1 and FGFR1 were identified and subsequently validated as targets for miR-149 (both the mature strand, miR-149, and the passenger strand, miR-149*) in endothelial cells (ECs). As a consequence of their targeting activity towards GPC1 and FGFR1, both miR-149 and miR-149* regulated FGF2 signaling and FGF2-induced responses in ECs, namely proliferation, migration and cord formation. Moreover, lentiviral overexpression of miR-149 reduced in vivo tumor-induced neovascularization. Importantly, FGF2 transcriptionally stimulated the expression of miR-149 independently of its host gene, therefore assuring the steady state of FGF2-induced responses through the regulation of the GPC1-FGFR1 binary complex in ECs.


Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Glipicanas/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/fisiologia , Neovascularização Fisiológica , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Células Cultivadas , Expressão Gênica , Glipicanas/metabolismo , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Interferência de RNA , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais
17.
Circ Res ; 112(12): 1592-601, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23519695

RESUMO

RATIONALE: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including adenosine triphosphate-binding cassette transporter A1 (ABCA1) and adenosine triphosphate-binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. OBJECTIVE: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. METHODS AND RESULTS: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed an miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to apolipoproteinA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 seems to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. CONCLUSIONS: miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression and modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease.


Assuntos
HDL-Colesterol/sangue , Hepatócitos/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteína A-I/metabolismo , Células COS , Chlorocebus aethiops , Dieta Hiperlipídica , Perfilação da Expressão Gênica/métodos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Homeostase , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/metabolismo , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Sulfonamidas/farmacologia
18.
J Exp Med ; 210(2): 301-19, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23359070

RESUMO

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Receptores Notch/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Modelos Animais de Doenças , Inativação Gênica , Homeostase , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ligantes , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/agonistas , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo
19.
Cell ; 149(1): 63-74, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22464323

RESUMO

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.


Assuntos
Eritropoese , Eritropoetina/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Anemia/prevenção & controle , Animais , Células Precursoras Eritroides/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/metabolismo , Camundongos , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
20.
J Bone Miner Res ; 27(3): 596-609, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22162090

RESUMO

Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1α and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.


Assuntos
Cartilagem/fisiologia , Sobrevivência Celular/fisiologia , Condrócitos/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Consumo de Oxigênio/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Apoptose , Cartilagem/citologia , Cartilagem/embriologia , Camundongos , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/genética
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