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1.
Am J Physiol Heart Circ Physiol ; 319(2): H306-H319, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32618513

RESUMO

Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal-regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.NEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.


Assuntos
Sinalização do Cálcio , Cardiomiopatia Dilatada/genética , Proteínas dos Microfilamentos/genética , Mutação , Contração Miocárdica , Miócitos Cardíacos/enzimologia , Fatores de Transcrição NFATC/metabolismo , Proteína Oncogênica v-akt/metabolismo , Função Ventricular Esquerda , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Células Cultivadas , Predisposição Genética para Doença , Cobaias , Masculino , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina I/genética , Troponina I/metabolismo , Troponina T/genética , Troponina T/metabolismo
2.
Int J Nanomedicine ; 15: 4705-4716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636626

RESUMO

Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease. Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot. Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs. Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.


Assuntos
Nanopartículas Metálicas/química , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Muramidase/química , Muramidase/metabolismo , Fatores de Transcrição NFATC/metabolismo , Nanomedicina/métodos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7
3.
Proc Natl Acad Sci U S A ; 117(28): 16638-16648, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601188

RESUMO

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição NFATC/genética , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Ligação Proteica , Transdução de Sinais , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
4.
Clin Sci (Lond) ; 134(12): 1305-1318, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32478397

RESUMO

Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Recent studies have demonstrated that the loss of Kruppel-like factor 15 (KLF15) in podocytes increases the susceptibility to injury; however, the mechanism underlying the protective effects on podocyte injury remains incompletely understood. Herein, we showed that KLF15 ameliorates podocyte injury through suppressing NFAT signaling and the salutary effects of the synthetic glucocorticoid dexamethasone in podocyte were partially mediated by the KLF15-NFATc1 axis. We found that KLF15 was significantly reduced in glomerular cells of proteinuric patients and in ADR-, LPS- or HG-treated podocyets in vitro. Overexpression of KLF15 attenuated podocyte apoptosis induced by ADR, LPS or HG and resulted in decreased expression of pro-apoptotic Bax and increased expression of anti-apoptotic Bcl-2. Conversely, the flow cytometry analysis and TUNEl assay demonstrated that loss of KLF15 accelerated podocyte apoptosis and we further found that 11R-VIVIT, a specific NFAT inhibitor, and NFATc1-siRNA rescued KLF15-deficient induced podocyte apoptosis. Meanwhile, Western blot and RT-qPCR showed that the expression of NFATc1 was up-regulated in KLF15 silenced podocytes and reduced in KLF15 overexpressed podocytes. Mechanistically, ChIP analysis showed that KLF15 bound to the NFATc1 promoter region -1984 to -1861base pairs upstream of the transcription start site and the binding amount was decreased after treatment with LPS. The dual-luciferase reporter assay indicated that NFATc1 was a direct target of KLF15. In addition, we found that in vitro treatment with dexamethasone induced a decrease of NFATc1 expression in podocytes and was abrogated by knockdown of KLF15. Hence, our results identify the critical role of the KLF15-NFATc1 axis in podocyte injury and loss, which may be involved in mediating the salutary effects of dexamethasone in podocytes.


Assuntos
Glucocorticoides/uso terapêutico , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição NFATC/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Glucose/toxicidade , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Biológicos , Transdução de Sinais
5.
Chem Biol Interact ; 327: 109179, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32534990

RESUMO

Excessive osteoclast leads to the imbalance in bone reconstruction and results in osteolytic diseases, such as osteoporosis and rheumatic arthritis. Integrin αvß3 abundantly expresses on osteoclast and plays a critical role in the formation and function of osteoclast, therefore, blockage of αvß3 has become an attractive therapeutic option for osteolytic diseases. In this study, we find that Tablysin-15, a RGD motif containing disintegrin, concentration-dependently suppresses RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption without affecting the cell viabilities. Tablysin-15 binds to integrin αvß3 and inhibits the activation of FAK-associated signaling pathways. Tablysin-15 also suppresses the activation of NF-кB, MAPK, and Akt-NFATc1 signaling pathways, which are crucial transcription factors during osteoclast differentiation. Moreover, Tablysin-15 decreases the osteoclastogenesis marker gene expression, including MMP-9, TRAP, CTSK, and c-Src. Finally, Tablysin-15 significantly inhibits LPS-induced bone loss in a mouse model. Taken together, our results indicate that Tablysin-15 significantly suppresses osteoclastogenesis in vitro and in vivo, thus it might be a excellent candidate for treating osteolytic-related diseases.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/prevenção & controle , Proteínas de Insetos/farmacologia , Osteogênese/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Animais , Conservadores da Densidade Óssea/toxicidade , Reabsorção Óssea/induzido quimicamente , Fêmur/efeitos dos fármacos , Fêmur/patologia , Proteínas de Insetos/toxicidade , Integrina alfaVbeta3/metabolismo , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Ligante RANK/metabolismo , Células RAW 264.7 , Proteínas e Peptídeos Salivares/toxicidade , Fator de Transcrição RelA/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
J Vasc Res ; 57(4): 185-194, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32526735

RESUMO

Information on the function of transient receptor potential vanilloid 1 (TRPV1) in arteriogenesis is limited. We aimed to verify whether TRPV1 is involved in collateral vessel growth in rat hind limbs and elucidate the possible subcellular action mechanisms. Adult Sprague Dawley rats were chosen to establish the hind limb ischemic model and treatment with capsaicin. Angiographies were performed, and tissue was isolated for immunohistochemistry. In vitro, rat aortic endothelial cells (RAECs) were treated with capsaicin and antagonist capsazepine. The RAEC proliferation was determined, and the protein and mRNA levels of Ca2+-dependent transcription factors were assessed. In vivo, the collateral vessels exhibited positive outward remodeling characterized by enhanced inflammatory cell/macrophage accumulation in the adventitia and activated cell proliferation in all layers of the vascular wall and elevated endothelial NO synthetase expression in the rats with hind limb ligation. In RAECs, TRPV1 activation-induced Ca2+-dependent transcriptional factors, nuclear factor of activated T cells 1, calsenilin and myocyte enhancer factor 2C increase, and augmented RAEC proliferation could be a subcellular mechanism for TRPV1 in endothelial cells and ultimately contribute to collateral vessel growth. TRPV1, a novel candidate, positively regulates arteriogenesis, meriting further studies to unravel the potential therapeutic target leading to improved collateral vessel growth for treating ischemic diseases.


Assuntos
Indutores da Angiogênese/farmacologia , Artérias/efeitos dos fármacos , Capsaicina/farmacologia , Circulação Colateral/efeitos dos fármacos , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Canais de Cátion TRPV/agonistas , Animais , Artérias/metabolismo , Artérias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Membro Posterior , Isquemia/metabolismo , Isquemia/fisiopatologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Fatores de Transcrição MEF2/metabolismo , Fatores de Transcrição NFATC/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Transdução de Sinais , Canais de Cátion TRPV/metabolismo
7.
Nat Med ; 26(6): 952-963, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32514169

RESUMO

In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.


Assuntos
Apolipoproteína E4/genética , Barreira Hematoencefálica/metabolismo , Calcineurina/metabolismo , Angiopatia Amiloide Cerebral/genética , Fatores de Transcrição NFATC/genética , Pericitos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Barreira Hematoencefálica/citologia , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição NFATC/metabolismo , Permeabilidade , RNA-Seq , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 11(1): 2444, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415068

RESUMO

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Imagem com Lapso de Tempo
9.
Phytomedicine ; 69: 153195, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32200293

RESUMO

BACKGROUND: Areca nut has anti-inflammatory, antiparasitic, antihypertensive, and antidepressant properties. The pathological hallmarks of inflammatory joint diseases are an increased number of osteoclasts and impaired differentiation of osteoblasts, which may disrupt the bone remodeling balance and eventually lead to bone loss. PURPOSE: The present study assessed the effects of arecoline, the main alkaloid found in areca nut, on osteoclast and osteoblast differentiation and function. METHOD: M-CSF/RANKL-stimulated murine bone marrow-derived macrophages (BMMs) were incubated with several concentrations of arecoline, and TRAP staining and pit formation were assessed to monitor osteoclast formation. Quantitative real-time RT-PCR and western blot analyses were used to analyze the expression of osteoclast-associated genes and signaling pathways. The effects of arecoline on bone were investigated in an in vivo mouse model of lipopolysaccharide (LPS)-induced trabecular bone loss after oral administration of arecoline. Alizarin red S staining and assays to measure ALP activity and the transcription level of osteoblast-related genes were used to evaluate the effects of arecoline on osteoblast differentiation and bone mineralization. RESULTS: In a dose-dependent manner, arecoline at concentrations of 50-100 µM reduced both the development of TRAP-positive multinucleated osteoclasts and the formation of resorption pits in M-CSF/RANKL-stimulated BMMs. In M-CSF/RANKL-stimulated BMMs, arecoline also suppressed the expression and translocation of c-Fos and NFATcl, and osteoclast differentiated-related genes via interference with the AKT, MAPK, and NF-kB activation pathways. Femur bone loss and microcomputed tomography parameters were recovered by oral administration of arecoline in the mouse LPS-induced bone loss model. Lastly, arecoline increased ALP activity, bone mineralization, and the expression of osteoblast differentiation-related genes, such as ALP and Runx2, in MC3T3-E1 cells. CONCLUSION: Our data suggest that arecoline may attenuate or prevent bone loss by suppressing osteoclastogenesis and promoting osteoblastogenesis. These findings provide evidence supporting arecoline's use as a potential therapeutic agent in bone-loss disorders and diseases.


Assuntos
Arecolina/farmacologia , Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos DBA , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Microtomografia por Raio-X
10.
Toxicology ; 436: 152429, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32156525

RESUMO

Excessive systemic uptake of inorganic fluorides causes disturbances of bone homeostasis. The mechanism of skeletal fluorosis is still uncertain. This study aimed to study the effect of fluoride on osteocyte-driven osteoclastogenesis and probe into the role of PTH in this process. IDG-SW3 cells seeded in collagen-coated constructs were developed into osteocyte-like cells through induction of mineral agents. Then, osteocyte-like cells were exposed to fluoride in the presence or absence of parathyroid hormone (PTH). Cell viability and their capacity to produce receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and sclerostin (SOST) were detected by MTT and Western blot assays, respectively. Finally, a transwell coculture system using osteocyte-like cells seeded in the low compartment, and osteoclast precursors added in the inserts was developed to observe the osteocyte-driven osteoclasogenesis response to fluoride with or without PTH, and the expression of molecules involved in this mechanism were measure by real time RT-PCR. Results showed that osteocytes withstood a toxic dose of fluoride, and yet PTH administration significantly reduced osteocytes viability. PTH amplified the effect of fluoride on the expression of osteoclastogenesis-related molecules in osteocyte, but did not enlarged the stimulating effect of fluoride on osteoclastogenesis drove by osteocyte coculture. Gene expression levels of TRAP, RANK, JNK and NFAtc1 significantly increased in fluoride affected osteoclast precursor cocultured with osteocyte-like cells. The impact of fluoride on osteocyte-driven osteoclast differentiation was stronger than that of PTH. In conclusion, osteocyte played a pivotal role on the mechanism underlying fluoride-affected osteoclastogenesis in which RANK-JNK-NFATc1 signaling pathway was involved, and PTH had a significant impact in this process.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fluoreto de Sódio/toxicidade , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteócitos/metabolismo , Osteócitos/patologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Hormônio Paratireóideo/farmacologia , Ligante RANK/genética , Ligante RANK/metabolismo , Células RAW 264.7 , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo
11.
Arterioscler Thromb Vasc Biol ; 40(5): 1256-1274, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32160773

RESUMO

OBJECTIVE: In view of our previous observations on differential expression of LMCD1 (LIM and cysteine-rich domains 1) in human versus rodents, we asked the question whether LMCD1 plays a species-specific role in the development of vascular lesions. Approach and Results: A combination of genetic, molecular, cellular, and disease models were used to test species-specific role of LMCD1 in the pathogenesis of vascular lesions. Here, we report species-specific regulation of LMCD1 expression in mediating vascular smooth muscle cell proliferation and migration during vascular wall remodeling in humans versus mice. Thrombin induced LMCD1 expression in human aortic smooth muscle cells but not mouse aortic smooth muscle cells via activation of Par1 (protease-activated receptor 1)-Gαq/11 (Gα protein q/11)-PLCß3 (phospholipase Cß3)-NFATc1 (nuclear factor of activated T cells 1) signaling. Furthermore, although LMCD1 mediates thrombin-induced proliferation and migration of both human aortic smooth muscle cells and mouse aortic smooth muscle cells via influencing E2F1 (E2F transcription factor 1)-mediated CDC6 (cell division cycle 6) expression and NFATc1-mediated IL (interleukin)-33 expression, respectively, in humans, it acts as an activator, and in mice, it acts as a repressor of these transcriptional factors. Interestingly, LMCD1 repressor activity was nullified by N-myristoyltransferase 2-mediated myristoylation in mouse. Besides, we found increased expression of LMCD1 in human stenotic arteries as compared to nonstenotic arteries. On the other hand, LMCD1 expression was decreased in neointimal lesions of mouse injured arteries as compared to noninjured arteries. CONCLUSIONS: Together, these observations reveal that LMCD1 acts as an activator and repressor of E2F1 and NFATc1 in humans and mice, respectively, in the induction of CDC6 and IL-33 expression during development of vascular lesions. Based on these findings, LMCD could be a potential target for drug development against restenosis and atherosclerosis in humans.


Assuntos
Proteínas Correpressoras/metabolismo , Fator de Transcrição E2F1/metabolismo , Interleucina-33/metabolismo , Proteínas com Domínio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Remodelação Vascular , Lesões do Sistema Vascular/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas Correpressoras/genética , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-33/genética , Proteínas com Domínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Ácido Mirístico/metabolismo , Fatores de Transcrição NFATC/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Especificidade da Espécie , Trombina/farmacologia , Remodelação Vascular/efeitos dos fármacos , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia
12.
Proc Natl Acad Sci U S A ; 117(14): 7961-7970, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209667

RESUMO

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.


Assuntos
Neoplasias da Mama/imunologia , Linfócitos do Interstício Tumoral/imunologia , MAP Quinase Quinase Quinases/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/transplante , Ciclofilina A/metabolismo , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Neoplasias Mamárias Experimentais/sangue , Neoplasias Mamárias Experimentais/patologia , Camundongos , Fatores de Transcrição NFATC/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Evasão Tumoral/efeitos dos fármacos
13.
Cell Prolif ; 53(3): e12774, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32034930

RESUMO

OBJECTIVES: Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. MATERIALS AND METHODS: Sprague Dawley rats were subjected to 28-day hindlimb-unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T-type CaV 3.1 channel was revealed by assessing its blocking effects. MiR-137 was identified as the upstream of CaV 3.1 channel by luciferase assay and investigated by gain/loss-of-function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV 3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. RESULTS: Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T-type CaV 3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR-137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T-type CaV 3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. CONCLUSIONS: The present work demonstrated that miR-137 and its target T-type CaV 3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.


Assuntos
Calcineurina/metabolismo , Canais de Cálcio Tipo T/metabolismo , Artérias Cerebrais/citologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Fatores de Transcrição NFATC/metabolismo , Animais , Encéfalo/irrigação sanguínea , Canais de Cálcio Tipo T/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Simulação de Ausência de Peso
14.
BMC Complement Med Ther ; 20(1): 35, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024503

RESUMO

BACKGROUND: Osteoporosis is related to the number and activity of osteoclasts. The goal of the present study was to demonstrate the effect of Chaenomelis Fructus (CF) on osteoclastogenesis and its mechanism of bone loss prevention in an OVX-induced osteoporosis model. METHODS: Osteoclasts were induced by RANKL in RAW 264.7 cells. TRAP assay was performed to measure the inhibitory effect of CF on osteoclast differentiation. Then, Expression of nuclear factor of activated T-cells (NFATc1), c-Fos which are essential transcription factors in osteoclastogenesis were detected using western blot and RT-PCR. The osteoclast-related markers were measured by RT-PCR. Moreover, the ability of CF to inhibit bone loss was researched by ovariectomized (OVX)-induced osteoporosis. RESULTS: Cell experiments showed that CF inhibited osteoclast differentiation and its function. Immunoblot analyses demonstrated that CF suppressed osteoclastogenesis through the NFATc1 and c-Fos signaling pathways. RT-PCR determined that CF inhibited osteoclast-related markers, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTK), osteoclast-associated immunoglobulin-like receptor (OSCAR), ATPase H+ Transporting V0 Subunit D2 (ATP6v0d2) and carbonic anhydrase II (CA2). In animal experiments, CF showed an inhibitory effect on bone density reduction through OVX. Hematoxylin and eosin (H&E) staining analysis data showed that CF inhibited OVX-induced trabecular area loss. TRAP staining and immunohistochemical staining analysis data showed that CF displayed an inhibitory effect on osteoclast differentiation through NFATc1 inhibition in femoral tissue. CONCLUSION: Based on the results of in vivo and in vitro experiments, CF inhibited the RANKL-induced osteoclasts differentiation and its function and effectively ameliorated OVX-induced osteoporosis rats.


Assuntos
Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Rosaceae/química , Animais , Western Blotting , Densidade Óssea , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fêmur/efeitos dos fármacos , Frutas/química , Camundongos , Osteoclastos/efeitos dos fármacos , Ovariectomia , Células RAW 264.7 , Ratos Sprague-Dawley
15.
FASEB J ; 34(2): 3197-3208, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31909857

RESUMO

Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1-c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1-c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN-binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1-dependent cytokine expression was downregulated. Through competition assays using NFATc1-derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275 ) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89 ) responsible for CNA/CNBR3 binding in which Cys263 and Asp82 , respectively, played crucial roles. The possible selective regulation of NFAT-mediated biological processes by targeting this new CN/NFAT-binding region is suggested.


Assuntos
Calcineurina/química , Simulação de Acoplamento Molecular , Fatores de Transcrição NFATC/química , Animais , Sítios de Ligação , Calcineurina/genética , Calcineurina/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Células Jurkat , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Ligação Proteica
16.
Int J Mol Sci ; 21(2)2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31947698

RESUMO

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.


Assuntos
Reabsorção Óssea/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/metabolismo , Animais , Catepsina K/genética , Catepsina K/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição NFATC/metabolismo , Especificidade de Órgãos , Ligante RANK/genética , Ligante RANK/metabolismo , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo
17.
FASEB J ; 34(2): 2392-2407, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908034

RESUMO

Bone resorption is a severe consequence of inflammatory diseases associated with osteolysis, such as rheumatoid arthritis (RA), often leading to disability in patients. In physiological conditions, the differentiation of bone-resorbing osteoclasts is delicately regulated by the balance between osteoclastogenic and anti-osteoclastogenic mechanisms. Inflammation has complex impact on osteoclastogenesis and bone destruction, and the underlying mechanisms of which, especially feedback inhibition, are underexplored. Here, we identify a novel regulatory network mediated by RBP-J/NFATc1-miR182 in TNF-induced osteoclastogenesis and inflammatory bone resorption. This network includes negative regulator RBP-J and positive regulators, NFATc1 and miR182, of osteoclast differentiation. In this network, miR182 is a direct target of both RBP-J and NFATc1. RBP-J represses, while NFATc1 activates miR182 expression through binding to specific open chromatin regions in the miR182 promoter. Inhibition of miR182 by RBP-J servers as a critical mechanism that limits TNF-induced osteoclast differentiation and inflammatory bone resorption. Inflammation, such as that which occurs in RA, shifts the expression levels of the components in this network mediated by RBP-J/NFATc1-miR182-FoxO3/PKR (previously identified miR182 targets) towards more osteoclastogenic, rather than healthy conditions. Treatment with TNF inhibitors in RA patients reverses the expression changes of the network components and osteoclastogenic potential. Thus, this network controls the balance between activating and repressive signals that determine the extent of osteoclastogenesis. These findings collectively highlight the biological significance and translational implication of this newly identified intrinsic regulatory network in inflammatory osteoclastogenesis and osteolysis.


Assuntos
Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteólise/metabolismo , Animais , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/patologia , Osteólise/genética , Osteólise/patologia
18.
Cancer Lett ; 473: 74-89, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904482

RESUMO

Gastrointestinal cancer causes countless deaths every year due to therapeutic resistance. However, whether metabolic alterations contribute to chemoresistance is not well understood. In this study, we report that fatty acid (FA) catabolism was activated in gastrointestinal cancer cells treated with oxaliplatin, which exhibited higher expression of the rate-limiting enzymes carnitine palmitoyltransferase 1B (CPT1B) and CPT2. The clinical analysis also showed that high expression of these enzymes was associated with poor oxaliplatin-based chemotherapy outcomes in patients. Furthermore, genetic or pharmacological inhibition of CPT2 with perhexiline disturbed NADPH and redox homeostasis and increased reactive oxygen species (ROS) generation and cell apoptosis in gastrointestinal cancer cells following oxaliplatin treatment. Specifically, the combination of oxaliplatin and perhexiline significantly suppressed the progression of gastrointestinal cancer in cell-based xenograft and patient-derived xenograft (PDX) models. Mechanistically, CPT2 was transcriptionally upregulated by nuclear factor of activated T cells 3 (NFATc3), which translocated to the nucleus in response to oxaliplatin treatment. In summary, our study suggests that the inhibition of CPT-mediated FA catabolism combined with conventional chemotherapy is a promising therapeutic strategy for patients with gastrointestinal cancers.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Ácidos Graxos/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , NADP/metabolismo , Fatores de Transcrição NFATC/metabolismo , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Perexilina/farmacologia , Perexilina/uso terapêutico , Espécies Reativas de Oxigênio , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
FASEB J ; 34(1): 1602-1619, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914620

RESUMO

Endurance exercise begun with reduced muscle glycogen stores seems to potentiate skeletal muscle protein abundance and gene expression. However, it is unknown whether this greater signaling responses is due to performing two exercise sessions in close proximity-as a first exercise session is necessary to reduce the muscle glycogen stores. In the present study, we manipulated the recovery duration between a first muscle glycogen-depleting exercise and a second exercise session, such that the second exercise session started with reduced muscle glycogen in both approaches but was performed either 2 or 15 hours after the first exercise session (so-called "twice-a-day" and "once-daily" approaches, respectively). We found that exercise twice-a-day increased the nuclear abundance of transcription factor EB (TFEB) and nuclear factor of activated T cells (NFAT) and potentiated the transcription of peroxisome proliferator-activated receptor-É£ coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-alpha (PPARα), and peroxisome proliferator-activated receptor beta/delta (PPARß/δ) genes, in comparison with the once-daily exercise. These results suggest that part of the elevated molecular signaling reported with previous "train-low" approaches might be attributed to performing two exercise sessions in close proximity. The twice-a-day approach might be an effective strategy to induce adaptations related to mitochondrial biogenesis and fat oxidation.


Assuntos
Biomarcadores/metabolismo , Exercício Físico/fisiologia , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adaptação Fisiológica/fisiologia , Adulto , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Estudos Cross-Over , Glicogênio/metabolismo , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fatores de Transcrição NFATC/metabolismo , Biogênese de Organelas , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
20.
Arterioscler Thromb Vasc Biol ; 40(3): 638-655, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31893948

RESUMO

OBJECTIVE: Although often studied independently, little is known about how aortic valve endothelial cells and valve interstitial cells interact collaborate to maintain tissue homeostasis or drive valve calcific pathogenesis. Inflammatory signaling is a recognized initiator of valve calcification, but the cell-type-specific downstream mechanisms have not been elucidated. In this study, we test how inflammatory signaling via NFκB (nuclear factor κ-light-chain enhancer of activated B cells) activity coordinates unique and shared mechanisms of valve endothelial cells and valve interstitial cells differentiation during calcific progression. Approach and Results: Activated NFκB was present throughout the calcific aortic valve disease (CAVD) process in both endothelial and interstitial cell populations in an established mouse model of hypercholesterolemia-induced CAVD and in human CAVD. NFκB activity induces endothelial to mesenchymal transformation in 3-dimensional cultured aortic valve endothelial cells and subsequent osteogenic calcification of transformed cells. Similarly, 3-dimensional cultured valve interstitial cells calcified via NFκB-mediated osteogenic differentiation. NFκB-mediated endothelial to mesenchymal transformation was directly demonstrated in vivo during CAVD via genetic lineage tracking. Genetic deletion of NFκB in either whole valves or valve endothelium only was sufficient to prevent valve-specific molecular and cellular mechanisms of CAVD in vivo despite the persistence of a CAVD inducing environment. CONCLUSIONS: Our results identify NFκB signaling as an essential molecular regulator for both valve endothelial and interstitial participation in CAVD pathogenesis. Direct demonstration of valve endothelial cell endothelial to mesenchymal transformation transmigration in vivo during CAVD highlights a new cellular population for further investigation in CAVD morbidity. The efficacy of valve-specific NFκB modulation in inhibiting hypercholesterolemic CAVD suggests potential benefits of multicell type integrated investigation for biological therapeutic development and evaluation for CAVD.


Assuntos
Valva Aórtica/metabolismo , Calcinose/metabolismo , Diferenciação Celular , Células Endoteliais/metabolismo , Doenças das Valvas Cardíacas/metabolismo , NF-kappa B/metabolismo , Osteogênese , Animais , Valva Aórtica/patologia , Calcinose/etiologia , Calcinose/patologia , Células Cultivadas , Microambiente Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Doenças das Valvas Cardíacas/etiologia , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/patologia , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
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