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1.
Proc Natl Acad Sci U S A ; 119(14): e2117112119, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35344430

RESUMO

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.


Assuntos
Colite , Fator de Transcrição STAT3 , Animais , Diferenciação Celular/genética , Colite/genética , Colite/metabolismo , Ativação Linfocitária , Camundongos , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Th17
2.
Arterioscler Thromb Vasc Biol ; 41(9): 2483-2493, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34320838

RESUMO

Objective: Despite considerable research, the goal of finding nonsurgical remedies against thoracic aortic aneurysm and acute aortic dissection remains elusive. We sought to identify a novel aortic PK (protein kinase) that can be pharmacologically targeted to mitigate aneurysmal disease in a well-established mouse model of early-onset progressively severe Marfan syndrome (MFS). Approach and Results: Computational analyses of transcriptomic data derived from the ascending aorta of MFS mice predicted a probable association between thoracic aortic aneurysm and acute aortic dissection development and the multifunctional, stress-activated HIPK2 (homeodomain-interacting protein kinase 2). Consistent with this prediction, Hipk2 gene inactivation significantly extended the survival of MFS mice by slowing aneurysm growth and delaying transmural rupture. HIPK2 also ranked among the top predicted PKs in computational analyses of DEGs (differentially expressed genes) in the dilated aorta of 3 MFS patients, which strengthened the clinical relevance of the experimental finding. Additional in silico analyses of the human and mouse data sets identified the TGF (transforming growth factor)-ß/Smad3 signaling pathway as a potential target of HIPK2 in the MFS aorta. Chronic treatment of MFS mice with an allosteric inhibitor of HIPK2-mediated stimulation of Smad3 signaling validated this prediction by mitigating thoracic aortic aneurysm and acute aortic dissection pathology and partially improving aortic material stiffness. Conclusions: HIPK2 is a previously unrecognized determinant of aneurysmal disease and an attractive new target for antithoracic aortic aneurysm and acute aortic dissection multidrug therapy.


Assuntos
Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Dissecção Aórtica/prevenção & controle , Fibrilina-1/genética , Síndrome de Marfan/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Remodelação Vascular/efeitos dos fármacos , Adulto , Dissecção Aórtica/enzimologia , Dissecção Aórtica/genética , Dissecção Aórtica/patologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/enzimologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Síndrome de Marfan/complicações , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Proteína Smad3/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 38(3): 588-591, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29371244

RESUMO

OBJECTIVE: Two were the aims of this study: first, to translate whole-genome expression profiles into computational predictions of functional associations between signaling pathways that regulate aorta homeostasis and the activity of angiotensin II type 1a receptor (At1ar) in either vascular endothelial or smooth muscle cells; and second, to characterize the impact of endothelial cell- or smooth muscle cell-specific At1ar disruption on the development of thoracic aortic aneurysm in fibrillin-1 hypomorphic (Fbn1mgR/mgR ) mice, a validated animal model of early onset progressively severe Marfan syndrome. APPROACH AND RESULTS: Cdh5-Cre and Sm22-Cre transgenic mice were used to inactivate the At1ar-coding gene (Agt1ar) in either intimal or medial cells of both wild type and Marfan syndrome mice, respectively. Computational analyses of differentially expressed genes predicted dysregulated signaling pathways of cell survival and matrix remodeling in Agt1arCdh5-/- aortas and of cell adhesion and contractility in Agt1arSm22-/- aortas. Characterization of Fbn1mgR/mgR;Agt1arCdh5-/- mice revealed increased median survival associated with mitigated aneurysm growth and media degeneration, as well as reduced levels of phosphorylated (p-) Erk1/2 but not p-Smad2. By contrast, levels of both p-Erk1/2 and p-Smad2 proteins were normalized in Fbn1mgR/mgR;Agt1arSm22-/- aortas in spite of them showing no appreciable changes in thoracic aortic aneurysm pathology. CONCLUSIONS: Physiological At1ar signaling in the intimal and medial layers is associated with distinct regulatory processes of aorta homeostasis and function; improper At1ar activity in the vascular endothelium is a significant determinant of thoracic aortic aneurysm development in Marfan syndrome mice.


Assuntos
Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/fisiopatologia , Biologia Computacional , Dilatação Patológica , Modelos Animais de Doenças , Células Endoteliais/patologia , Fibrilina-1/genética , Fibrilina-1/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Homeostase , Masculino , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Receptor Tipo 1 de Angiotensina/deficiência , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais
4.
Blood ; 125(8): e1-13, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25573988

RESUMO

Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated messenger RNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R-regulated miR-21 network that modulates macrophage polarization.


Assuntos
Inflamação/genética , Macrófagos Peritoneais/imunologia , MicroRNAs/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Polaridade Celular/genética , Polaridade Celular/imunologia , Células Cultivadas , Redes Reguladoras de Genes , Inflamação/imunologia , Ativação de Macrófagos/genética , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Regulação para Cima/genética , Regulação para Cima/imunologia
5.
Cancer Discov ; 13(6): 1386-1407, 2023 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-37061969

RESUMO

Predicting in vivo response to antineoplastics remains an elusive challenge. We performed a first-of-kind evaluation of two transcriptome-based precision cancer medicine methodologies to predict tumor sensitivity to a comprehensive repertoire of clinically relevant oncology drugs, whose mechanism of action we experimentally assessed in cognate cell lines. We enrolled patients with histologically distinct, poor-prognosis malignancies who had progressed on multiple therapies, and developed low-passage, patient-derived xenograft models that were used to validate 35 patient-specific drug predictions. Both OncoTarget, which identifies high-affinity inhibitors of individual master regulator (MR) proteins, and OncoTreat, which identifies drugs that invert the transcriptional activity of hyperconnected MR modules, produced highly significant 30-day disease control rates (68% and 91%, respectively). Moreover, of 18 OncoTreat-predicted drugs, 15 induced the predicted MR-module activity inversion in vivo. Predicted drugs significantly outperformed antineoplastic drugs selected as unpredicted controls, suggesting these methods may substantively complement existing precision cancer medicine approaches, as also illustrated by a case study. SIGNIFICANCE: Complementary precision cancer medicine paradigms are needed to broaden the clinical benefit realized through genetic profiling and immunotherapy. In this first-in-class application, we introduce two transcriptome-based tumor-agnostic systems biology tools to predict drug response in vivo. OncoTarget and OncoTreat are scalable for the design of basket and umbrella clinical trials. This article is highlighted in the In This Issue feature, p. 1275.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transcriptoma , Medicina de Precisão/métodos , Oncologia/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico
6.
Biochem J ; 424(1): 79-88, 2009 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-19715556

RESUMO

The metalloproteinases TACE [tumour necrosis factor alpha-converting enzyme; also known as ADAM17 (a disintegrin and metalloproteinase 17)] and ADAM10 are the primary enzymes responsible for catalysing release of membrane-anchored proteins from the cell surface in metazoan organisms. Although the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1' position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, whereas ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identified three residues in the S1' pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites.


Assuntos
Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Sítios de Ligação/genética , Domínio Catalítico/genética , Linhagem Celular , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese , Ligação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Necrose Tumoral/metabolismo
7.
JCI Insight ; 4(11)2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31167969

RESUMO

Marfan syndrome (MFS) is associated with mutations in fibrillin-1 that predispose afflicted individuals to progressive thoracic aortic aneurysm (TAA) leading to dissection and rupture of the vessel wall. Here we combined computational and experimental approaches to identify and test FDA-approved drugs that may slow or even halt aneurysm progression. Computational analyses of transcriptomic data derived from the aortas of MFS patients and MFS mice (Fbn1mgR/mgR mice) predicted that subcellular pathways associated with reduced muscle contractility are key TAA determinants that could be targeted with the GABAB receptor agonist baclofen. Systemic administration of baclofen to Fbn1mgR/mgR mice validated our computational prediction by mitigating arterial disease progression at the cellular and physiological levels. Interestingly, baclofen improved muscle contraction-related subcellular pathways by upregulating a different set of genes than those downregulated in the aorta of vehicle-treated Fbn1mgR/mgR mice. Distinct transcriptomic profiles were also associated with drug-treated MFS and wild-type mice. Thus, systems pharmacology approaches that compare patient- and mouse-derived transcriptomic data for subcellular pathway-based drug repurposing represent an effective strategy to identify potential new treatments of human diseases.


Assuntos
Aneurisma da Aorta Torácica , Reposicionamento de Medicamentos/métodos , Transcriptoma/efeitos dos fármacos , Animais , Aneurisma da Aorta Torácica/tratamento farmacológico , Aneurisma da Aorta Torácica/etiologia , Aneurisma da Aorta Torácica/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Síndrome de Marfan/complicações , Camundongos , Camundongos Transgênicos
8.
J Bacteriol ; 186(19): 6515-25, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15375133

RESUMO

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.


Assuntos
Bifidobacterium/metabolismo , Frutoquinases/fisiologia , Frutose/metabolismo , Catálise , Frutoquinases/genética , Glucose/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transcrição Gênica
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