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1.
J Am Heart Assoc ; 10(16): e020554, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34350769

RESUMO

Background Adventitial remodeling is a pathological hallmark of hypertension that results in target organ damage. Activated adventitial fibroblasts have emerged as critical regulators in this process, but the precise mechanism remains unclear. Methods and Results Interleukin 11 (IL-11) knockout and wild-type mice were subjected to angiotensin II (Ang II) infusion to establish models of hypertension-associated vascular remodeling. IL-11 mRNA and protein were increased especially in the adventitia in response to Ang II. Compared with wild-type mice, Ang II-treated IL-11 knockout mice showed amelioration of vascular hypertrophy, adventitial fibrosis, macrophage infiltration, and inflammatory factor expression. Recombination mouse IL-11 exacerbated adventitial fibrosis in Ang II-infused wild-type mice. Interestingly, IL-11 neutralizing antibody attenuated adventitial fibrosis, macrophage infiltration, and inflammatory factor expression after Ang II infusion for 7 days. Mechanistically, in primary cultured adventitial fibroblasts, Krüppel-like factor 15 negatively regulated Ang II-induced IL-11 expression. Ang II increased extracellular signal-regulated kinases 1 and 2 activation, especially in adventitia, and caused biphasic extracellular signal-regulated kinases 1 and 2 activation in adventitial fibroblasts. A rapid and early activation increased IL-11 production through decreasing Krüppel-like factor 15 expression, which, in turn, induced the second extracellular signal-regulated kinases 1 and 2 activation, resulting in posttranscriptional profibrotic gene expression. Conclusions These results demonstrate that extracellular signal-regulated kinases 1 and 2 activation is important for Krüppel-like factor 15-mediated IL-11 expression in adventitial fibroblasts to promote adventitial remodeling in Ang II-induced hypertension. Therefore, targeting the Krüppel-like factor 15/IL-11 axis might serve as a new therapeutic strategy for vascular diseases.


Assuntos
Túnica Adventícia/enzimologia , Aorta Torácica/enzimologia , Fibroblastos/enzimologia , Hipertensão/enzimologia , Interleucina-11/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Remodelação Vascular , Túnica Adventícia/patologia , Angiotensina II , Animais , Aorta Torácica/patologia , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Células HEK293 , Humanos , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/patologia , Mediadores da Inflamação/metabolismo , Interleucina-11/genética , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos Sprague-Dawley , Transdução de Sinais
2.
Cell Death Dis ; 12(7): 699, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262020

RESUMO

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.


Assuntos
Apoptose/efeitos dos fármacos , Hidroxianisol Butilado/farmacologia , Fibroblastos/efeitos dos fármacos , Aditivos Alimentares/farmacologia , Necroptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Animais , Antioxidantes/farmacologia , Modelos Animais de Doenças , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Ligação Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Fator de Necrose Tumoral alfa
3.
Nat Commun ; 12(1): 4359, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272378

RESUMO

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.


Assuntos
Fibroblastos/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Sistemas CRISPR-Cas , Sequenciamento de Cromatina por Imunoprecipitação , Cromatografia Líquida , Desmetilação , Epigênese Genética , Fibroblastos/enzimologia , Deleção de Genes , Heterocromatina/enzimologia , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histona-Lisina N-Metiltransferase/genética , Hibridização in Situ Fluorescente , Espectrometria de Massas , Metilação , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Processamento de Proteína Pós-Traducional/genética , RNA-Seq , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Transdução de Sinais/genética
4.
Front Immunol ; 12: 672461, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248953

RESUMO

Objectives: Psoriatic arthritis (PsA) is a chronic inflammatory disease associated with psoriasis. Janus Kinase inhibitors (JAKi) have emerged as an encouraging class of drugs for the treatment of PsA. Here, we compare the effect of four JAKi on primary PsA synovial fibroblasts (PsAFLS) activation, metabolic function, and invasive and migratory capacity. Methods: Primary PsAFLS were isolated and cultured with JAKi (Peficitinib, Filgotinib, Baricitinib and Upadacitinib) in the presence of Oncostatin M (OSM). pSTAT3 expression in response to OSM was quantified by Western Blot analysis. Pro-inflammatory cytokines/chemokines were quantified by ELISA and cell migration by wound-repair scratch assays. Invasive capacity was examined using Matrigel™ invasion chambers and MMP multiplex MSD assays. PsAFLS bioenergetics was assessed using the Seahorse XFe Extracellular Flux Analyzer, which simultaneously quantifies two energetic pathways- glycolysis (ECAR) and oxidative phosphorylation (OCR). In parallel, inflammatory, invasive, and migratory genes were quantified by RT-PCR. Results: OSM induces pSTAT3 expression in PsAFLS. OSM-induced secretion of MCP-1 and IL-6 was inhibited by all JAKi with Peficitinib, Baricitinib and Upadacitinib showing the greatest effect. In contrast, JAKi had no significant impact on IL-8 expression in response to OSM. PsAFLS cell invasion, migratory capacity and MMP1, 3, and 9 were suppressed following JAKi treatment, with Peficitinib showing the greatest effect. These functional effects were accompanied by a change in the cellular bioenergetic profile of PsAFLS, where JAKi significantly decreased glycolysis and the ECAR/OCR, resulting in a shift to a more quiescent phenotype, with Peficitinib demonstrating the most pronounced effect. Conclusion: This study demonstrates that JAK/STAT signalling mediates the complex interplay between inflammation and cellular metabolism in PsA pathogenesis. This inhibition shows effective suppression of inflammatory mechanisms that drive pathogenic functions of PsAFLS, further supporting the role of JAKi as a therapeutic target for the treatment of PsA.


Assuntos
Artrite Psoriásica , Fibroblastos/efeitos dos fármacos , Inibidores de Janus Quinases/farmacologia , Janus Quinases/antagonistas & inibidores , Fatores de Transcrição STAT/antagonistas & inibidores , Adamantano/análogos & derivados , Adamantano/farmacologia , Adulto , Idoso , Artrite Psoriásica/imunologia , Artrite Psoriásica/metabolismo , Azetidinas/farmacologia , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Membrana Sinovial/efeitos dos fármacos , Triazóis/farmacologia
5.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204949

RESUMO

Idiopathic pulmonary fibrosis (IPF) is one of the most symptomatic progressive fibrotic lung diseases, in which patients have an extremely poor prognosis. Therefore, understanding the precise molecular mechanisms underlying pulmonary fibrosis is necessary for the development of new therapeutic options. Stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) are ubiquitously expressed in various types of cells and activated in response to cellular environmental stresses, including inflammatory and apoptotic stimuli. Type II alveolar epithelial cells, fibroblasts, and macrophages are known to participate in the progression of pulmonary fibrosis. SAPKs can control fibrogenesis by regulating the cellular processes and molecular functions in various types of lung cells (including cells of the epithelium, interstitial connective tissue, blood vessels, and hematopoietic and lymphoid tissue), all aspects of which remain to be elucidated. We recently reported that the stepwise elevation of intrinsic p38 signaling in the lungs is correlated with a worsening severity of bleomycin-induced fibrosis, indicating an importance of this pathway in the progression of pulmonary fibrosis. In addition, a transcriptome analysis of RNA-sequencing data from this unique model demonstrated that several lines of mechanisms are involved in the pathogenesis of pulmonary fibrosis, which provides a basis for further studies. Here, we review the accumulating evidence for the spatial and temporal roles of SAPKs in pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase 4/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/crescimento & desenvolvimento , Fibroblastos/enzimologia , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/patologia , Pulmão/embriologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/enzimologia
6.
Aging (Albany NY) ; 13(12): 15750-15769, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34102611

RESUMO

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated ß-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.


Assuntos
Ceramidase Ácida/metabolismo , Senescência Celular , Fibroblastos/citologia , Fibroblastos/enzimologia , Ceramidase Ácida/genética , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular , Ceramidas/metabolismo , Inativação Gênica , Humanos , Metaboloma , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética
7.
Cells ; 10(5)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069977

RESUMO

Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.


Assuntos
Ácidos Graxos/metabolismo , Fibroblastos/enzimologia , Erros Inatos do Metabolismo Lipídico/enzimologia , Lipidômica , Metaboloma , Pele/enzimologia , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Estudos de Casos e Controles , Células Cultivadas , Ceramidas/metabolismo , Feminino , Humanos , Erros Inatos do Metabolismo Lipídico/genética , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/deficiência , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/genética , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Oxirredução , Esfingolipídeos/metabolismo , Espectrometria de Massas em Tandem
8.
Hum Genet ; 140(7): 1077-1096, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33944995

RESUMO

The Okur-Chung neurodevelopmental syndrome, or OCNDS, is a newly discovered rare neurodevelopmental disorder. It is characterized by developmental delay, intellectual disability, behavioral problems (hyperactivity, repetitive movements and social interaction deficits), hypotonia, epilepsy and language/verbalization deficits. OCNDS is linked to de novo mutations in CSNK2A1, that lead to missense or deletion/truncating variants in the encoded protein, the protein kinase CK2α. Eighteen different missense CK2α mutations have been identified to date; however, no biochemical or cell biological studies have yet been performed to clarify the functional impact of such mutations. Here, we show that 15 different missense CK2α mutations lead to varying degrees of loss of kinase activity as recombinant purified proteins and when mutants are ectopically expressed in mammalian cells. We further detect changes in the phosphoproteome of three patient-derived fibroblast lines and show that the subcellular localization of CK2α is altered for some of the OCNDS-linked variants and in patient-derived fibroblasts. Our data argue that reduced kinase activity and abnormal localization of CK2α may underlie the OCNDS phenotype.


Assuntos
Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Animais , Células COS , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular , Chlorocebus aethiops , Fibroblastos/enzimologia , Humanos , Espectrometria de Massas , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação de Sentido Incorreto
9.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34037658

RESUMO

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.


Assuntos
Senescência Celular , Metilação de DNA , Epigênese Genética , Fibroblastos/enzimologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Interleucina-1alfa/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Feminino , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Interleucina-1alfa/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia de Fluorescência , Papiloma/induzido quimicamente , Papiloma/genética , Papiloma/metabolismo , Papiloma/patologia , Fenótipo , Via Secretória , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol
10.
Cells ; 10(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799608

RESUMO

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.


Assuntos
Colágeno/metabolismo , Fibroblastos/enzimologia , Pulmão/enzimologia , Fibrose Pulmonar/enzimologia , Adulto , Animais , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/patologia , Deleção de Genes , Predisposição Genética para Doença , Humanos , Interleucina-18/genética , Interleucina-18/metabolismo , Pulmão/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Pessoa de Meia-Idade , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Osteopontina/genética , Osteopontina/metabolismo , Fenótipo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais
11.
Cell Death Dis ; 12(3): 249, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674551

RESUMO

TG2 is a multifunctional enzyme involved in several cellular processes and has emerging as a potential regulator of gene expression. In this regard, we have recently shown that TG2 is able to activate HSF1, the master transcriptional regulator of the stress-responsive genes; however, its effect on the overall gene expression remains unclear. To address this point, we analyzed, by RNA-seq, the effect of TG2 on the overall transcriptome as well as we characterized the TG2 interactome in the nucleus. The data obtained from these omics approaches reveal that TG2 markedly influences the overall cellular transcriptome profile and specifically the Wnt and HSF1 pathways. In particular, its ablation leads to a drastic downregulation of many key members of these pathways. Interestingly, we found that key components of the Wnt/ß-catenin pathway are also downregulated in cells lacking HSF1, thus confirming that TG2 regulates the HSF1 and this axis controls the Wnt signaling. Mechanistic studies revealed that TG2 can regulate the Wnt pathway by physically interacts with ß-catenin and its nuclear interactome includes several proteins known to be involved in the regulation of the Wnt signaling. In order to verify whether this effect is playing a role in vivo, we ablated TG2 in Danio rerio. Our data show that the zebrafish lacking TG2 cannot complete the development and their death is associated with an evident downregulation of the Wnt pathway and a defective heat-shock response. Our findings show for the first time that TG2 is essential for the correct embryonal development of lower vertebrates, and its action is mediated by the Wnt/HSF1 axis.


Assuntos
Fibroblastos/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Transglutaminases/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transcrição Genética , Transcriptoma , Transglutaminases/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
12.
Biochem Biophys Res Commun ; 549: 34-39, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33662666

RESUMO

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.


Assuntos
Citocinas/farmacologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Pentosiltransferases/biossíntese , Escleroderma Sistêmico/enzimologia , Pele/patologia , Indução Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Pentosiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta1/farmacologia
13.
Am J Pathol ; 191(5): 857-871, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33640318

RESUMO

To investigate the role of glycolysis and the M2 isoform of pyruvate kinase (PKM2) in odontogenic keratocysts (OKCs), the glycolytic flux of primary odontogenic keratocyst fibroblasts (OKC-Fs) and normal oral mucosa fibroblasts (OM-Fs) was determined by glucose uptake, lactate production, and cell proliferation assays. Wound healing assay and Matrigel-coated chamber system were used to investigate the effects of PKM2 on migration and invasion capacities of OKC-Fs. Co-culture of OKC-Fs with osteoclast precursors (RAW264.7 cells) was used to clarify the role of glycolysis in the osteoclastogenic effects of OKC-Fs. In addition, hypoxia-inducible factor 1α and some key enzymes related to glycolysis, including PKM2, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A, were detected to assess the activation of glycolysis in OKC stroma by immunohistochemistry. Results showed that the glucose uptake and lactate production were significantly higher in OKC-Fs than OM-Fs. PKM2 was elevated in OKC-Fs compared with that in OM-Fs. PKM2 significantly regulated glycolysis, proliferation, migration, invasion, and osteoclastogenic effects of OKC-Fs. Additionally hypoxia-inducible factor 1α, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A were markedly overexpressed in OKC stroma, and correlated with PKM2. Moreover, the expression of PKM2 was regulated by oxygen concentration in vitro. In sum, PKM2-mediated glycolysis regulated the growth, aggressiveness, and osteoclastogenesis of OKC.


Assuntos
Glicólise , Cistos Odontogênicos/enzimologia , Osteogênese , Piruvato Quinase/metabolismo , Animais , Movimento Celular , Proliferação de Células , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Camundongos , Invasividade Neoplásica , Cistos Odontogênicos/patologia , Oxigênio/metabolismo , Isoformas de Proteínas , Piruvato Quinase/genética , Células RAW 264.7
14.
Eur J Cancer ; 147: 63-73, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33618200

RESUMO

OBJECTIVE: The mechanisms underlying the contribution of primary tumour to pre-metastatic niche formation remains largely unknown in hepatocellular carcinoma (HCC). We previously reported that the released LOXL2 from HCC cells under higher stiffness stimulation facilitated the formation of lung pre-metastatic niche. Here, we further clarified the pathological role of LOXL2 in promoting lung pre-metastatic niche formation and lung metastasis occurrence in HCC and its relevant molecular mechanism. METHODS: Using two different animal models and an in vitro system of mechanically tuneable gel mirroring lung tissue stiffness, we explored the underlying mechanism of LOXL2 in pre-metastatic niche formation. RESULTS: We applied tail vein injection of CM-LV-LOXL2-OEsimulating tumour-released soluble factors to induce lung pre-metastatic niche formation and found that the injected LOXL2 remarkably enhanced CD11b+/CD45+ bone marrow-derived cells (BMDCs) recruitment and fibronectin expression in lung. Subsequently, LOXL2-overexpressed xenograft HCC models validated that tumour-secreted LOXL2 significantly promoted the occurrence of pulmonary metastasis. In vitro, LOXL2 and LOXL2-caused matrix stiffening not only obviously upregulated the expressions of MMP9 and fibronectin in lung fibroblasts, but also evidently increased the number of adherent HCC cells and the expression of chemokine CXCL12. The activation of PI3K-AKT pathway mediated LOXL2-upregulated fibronectin. HCC patients in High-LOXL2 group had higher ratio of tumour recurrence than HCC patients in Low-LOXL2 group, supporting a significance of LOXL2 in HCC progression and unfavourable outcome. CONCLUSION: Primary tumour-released LOXL2 promotes lung pre-metastatic niche formation and lung metastasis occurrence. LOXL2-caused matrix stiffening synergistically regulates lung pre-metastatic niche formation. Targeting LOXL2-induced lung pre-metastatic niche may be a novel intervention approach against HCC metastasis.


Assuntos
Aminoácido Oxirredutases/metabolismo , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Pulmonares/enzimologia , Microambiente Tumoral , Aminoácido Oxirredutases/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
15.
Cell Death Dis ; 12(2): 172, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568640

RESUMO

As evidenced by the behavior of loss-of-function mutants of PTEN in the context of a gain-of-function mutation of AKT1, the PTEN-AKT1 signaling pathway plays a critical role in human cancers. In this study, we demonstrated that a deficiency in PTEN or activation of AKT1 potentiated the expression of platelet-derived growth factor receptor α (PDGFRα) based on studies on Pten-/- mouse embryonic fibroblasts, human cancer cell lines, the hepatic tissues of Pten conditional knockout mice, and human cancer tissues. Loss of PTEN enhanced PDGFRα expression via activation of the AKT1-CREB signaling cascade. CREB transactivated PDGFRα expression by direct binding of the promoter of the PDGFRα gene. Depletion of PDGFRα attenuated the tumorigenicity of Pten-null cells in nude mice. Moreover, the PI3K-AKT signaling pathway has been shown to positively correlate with PDGFRα expression in multiple cancers. Augmented PDGFRα was associated with poor survival of cancer patients. Lastly, combination treatment with the AKT inhibitor MK-2206 and the PDGFR inhibitor CP-673451 displayed synergistic anti-tumor effects. Therefore, activation of the AKT1-CREB-PDGFRα signaling pathway contributes to the tumor growth induced by PTEN deficiency and should be targeted for cancer treatment.


Assuntos
Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fibroblastos/enzimologia , Fígado/enzimologia , Neoplasias/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , PTEN Fosfo-Hidrolase/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Quinolinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Transplantation ; 105(6): 1212-1224, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33560725

RESUMO

BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.


Assuntos
Bronquiolite Obliterante/enzimologia , Fibroblastos/enzimologia , Transplante de Pulmão/efeitos adversos , Pulmão/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Adolescente , Adulto , Idoso , Animais , Bronquiolite Obliterante/tratamento farmacológico , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/patologia , Proliferação de Células , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , França , Humanos , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/cirurgia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Reepitelização , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima , Adulto Jovem
17.
Biol Pharm Bull ; 44(1): 131-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33390540

RESUMO

Rheumatoid arthritis (RA) is an inflammatory disease with joint dysfunction following cartilage degradation. The level of lysophosphatidic acid (LPA) has been reported to be augmented in human synovial fluid from patients with RA. However, it remains to be elucidated whether LPA participates in cartilage destruction. In the present study, we have demonstrated that the production of promatrix metalloproteinases (proMMPs)-1 and -3 was augmented along with an increase of extracellular signal-regulated kinase (ERK)1/2 phosphorylation through LPA receptor 1 (LPAR1) in human synovial fibroblasts. These results suggest that LPA transcriptionally increases MMP production by the activation of an LPAR1/ERK1/2 signal pathway in human synovial fibroblasts. Thus, LPA is likely to be a pathological candidate for cartilage degradation in RA.


Assuntos
Fibroblastos/enzimologia , Lisofosfolipídeos/farmacologia , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 3 da Matriz/biossíntese , Membrana Sinovial/enzimologia , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Membrana Sinovial/efeitos dos fármacos
18.
Biol Pharm Bull ; 44(1): 18-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33390545

RESUMO

Deeper wrinkles and loss of elasticity are one of the skin-aging symptoms. Collagen breakdown by matrix metalloproteinase-1 (MMP-1), which is induced by reactive oxygen species (ROS) and pro-inflammatory cytokines, has been known to be responsible for these skin-aging symptoms. Therefore, much attention has been paid to chemicals to suppress the MMP-1 activity. Epigallocatechin-3-gallate (EGCG), catechin rich in green tea, has been reported to show antioxidant and protect skin from various stimuli such as UV and chemicals. In this study, we evaluated the inhibitory effect of EGCG on MMP-1 gene expression and secretion in tumor necrosis factor-α (TNF-α)-treated human dermal fibroblast cells (Hs68 cells). Pre-treatment with EGCG (10 and 20 µM) suppressed TNF-α-induced MMP-1 expression and secretion. EGCG also reduced the phosphorylation of extracellular signal regulated kinase (ERK) significantly but not that of p38 activation and c-Jun N-terminal kinase (JNK). Besides, EGCG (10 and 20 µM) showed the inhibitory effect on mitogen-activated protein extracellular kinase (MEK) and Src phosphorylation which is reported to be upstream signal proteins of ERK signal pathway. Based on these results, EGCG might have potential activity to slow down the skin-aging through inhibition of collagen breakdown, which remains to be elucidated.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Fibroblastos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 1 da Matriz/biossíntese , Fator de Necrose Tumoral alfa/toxicidade , Catequina/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
19.
Sci Rep ; 11(1): 2157, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495511

RESUMO

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.


Assuntos
Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo , RecQ Helicases/deficiência , Autofagia , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Fibroblastos/enzimologia , Fibroblastos/patologia , Fase G1 , Humanos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Mitose , Espécies Reativas de Oxigênio/metabolismo , RecQ Helicases/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima
20.
Arterioscler Thromb Vasc Biol ; 41(2): 698-710, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33054395

RESUMO

OBJECTIVE: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. CONCLUSIONS: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.


Assuntos
Fibroblastos/enzimologia , Hipertensão/enzimologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidase 2/metabolismo , Comunicação Parácrina , Remodelação Vascular , Angiotensina II , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Pressão Sanguínea , Células Cultivadas , Modelos Animais de Doenças , Fator 6 de Diferenciação de Crescimento/genética , Fator 6 de Diferenciação de Crescimento/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , NADPH Oxidase 2/genética , Transdução de Sinais
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