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1.
Artigo em Inglês | MEDLINE | ID: mdl-38861338

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline due to scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial to mesenchymal transition and oxidative stress. The RhoA/ROCK signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in IPF patients and is a promising target for IPF therapy. However, due to hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ~80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain (p-MLC) and reduced the expression of fibrosis-related proteins, such as collagen, fibronectin, and alpha-smooth muscle actin (α-SMA) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor beta (TGF-ß)-induced fibroblast-to-myofibroblast transition (FMT). Additionally, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

2.
Int J Mol Sci ; 25(12)2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38928356

RESUMO

The topology of the basement membrane (BM) affects cell physiology and pathology, and BM thickening is associated with various chronic lung diseases. In addition, the topology of commercially available poly (ethylene terephthalate) (PET) membranes, which are used in preclinical in vitro models, differs from that of the human BM, which has a fibrous and elastic structure. In this study, we verified the effect of BM thickness on the differentiation of normal human bronchial epithelial (NHBE) cells. To evaluate whether the thickness of poly-ε-carprolactone (PCL) mesh affects the differentiation of NHBE cells, cells were grown on thin- (6-layer) and thick-layer (80-layer) meshes consisting of electrospun PCL nanofibers using an air-liquid interface (ALI) cell culture system. It was found that the NHBE cells formed a normal pseudostratified epithelium composed of ciliated, goblet, and basal cells on the thin-layer PCL mesh; however, goblet cell hyperplasia was observed on the thick-layer PCL mesh. Differentiated NHBE cells cultured on the thick-layer PCL mesh also demonstrated increased epithelial-mesenchymal transition (EMT) compared to those cultured on the thin-layer PCL mesh. In addition, expression of Sox9, nuclear factor (NF)-κB, and oxidative stress-related markers, which are also associated with goblet cell hyperplasia, was increased in the differentiated NHBE cells cultured on the thick-layer PCL mesh. Thus, the use of thick electrospun PCL mesh led to NHBE cells differentiating into hyperplastic goblet cells via EMT and the oxidative stress-related signaling pathway. Therefore, the topology of the BM, for example, thickness, may affect the differentiation direction of human bronchial epithelial cells.


Assuntos
Membrana Basal , Diferenciação Celular , Células Epiteliais , Poliésteres , Humanos , Poliésteres/química , Membrana Basal/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Nanofibras/química , Células Cultivadas , Brônquios/citologia , Brônquios/metabolismo
3.
BMC Mol Cell Biol ; 23(1): 55, 2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517746

RESUMO

Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1ß expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.


Assuntos
Cardiotoxicidade , Piroptose , Humanos , Piroptose/fisiologia , Cardiotoxicidade/metabolismo , Inflamassomos/metabolismo , Inflamassomos/farmacologia , Miócitos Cardíacos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/farmacologia , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/farmacologia , NF-kappa B/metabolismo , Transdução de Sinais , Doxorrubicina/efeitos adversos , Doxorrubicina/metabolismo
4.
Front Cell Dev Biol ; 9: 652791, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33768098

RESUMO

Spinophilin (SPL) is a multifunctional actin-binding scaffolding protein. Although increased research on SPL in cancer biology has revealed a tumor suppressive role, its modulation in cancer biology, and oncological relevance remains elusive. Thus, we determined the role of SPL in the modulation of the junctional network and cellular migration in A549 lung cancer cell line. Knockdown of SPL promoted cancer cell invasion in agarose spot and scratch wound assays. Attenuation of SPL expression also enhanced invadopodia, as revealed by enhanced vinculin spots, and enhanced sodium bicarbonate cotransporter NBC activity without enhancing membranous expression of NBCn1. Disruption of the tubular structure with nocodazole treatment revealed enhanced SPL expression and reduced NBC activity and A549 migration. SPL-mediated junctional modulation and tubular stability affected bicarbonate transporter activity in A549 cells. The junctional modulatory function of SPL in start-up migration, such as remodeling of tight junctions, enhanced invadopodia, and increased NBC activity, revealed here would support fundamental research and the development of an initial target against lung cancer cell migration.

5.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182643

RESUMO

Keratinocyte migration is initiated toward the wound skin barrier as a crucial process in wound healing. However, the migratory machinery used by keratinocytes is relatively unknown. Histamine signaling, including an increase in the Ca2+ signal, mediated the enhanced protein expression and chloride/bicarbonate exchange activity of anion exchanger AE2 in keratinocytes. In this study, we applied an agarose spot assay to induce a vectorial motion. The vectorial stimulation of the histamine-containing agarose spot enhanced the HaCaT keratinocyte migration, compared to non-directional stimulation. AE2 is associated with the vectorial movement of HaCaT keratinocytes. Enhanced expression of AE2 was mainly associated with an increase in Ca2+ and was abolished by the treatment with the Ca2+ chelating agent BAPTA-AM. These findings revealed that the directionality of Ca2+-exerted stimulation can play a prominent role in facilitating migration through the involvement of AE2 as a migratory machinery in HaCaT keratinocytes.


Assuntos
Sinalização do Cálcio , Queratinócitos/fisiologia , Cloreto de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Dissulfiram/farmacologia , Histamina/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Modelos Biológicos , Pele/lesões , Pele/patologia , Pele/fisiopatologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
6.
Pharmaceutics ; 12(9)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867284

RESUMO

IP3 receptor-binding protein released with IP3 (IRBIT) interacts with various ion channels and transporters. An electroneutral type of sodium bicarbonate cotransporter, NBCn1, participates in cell migration, and its enhanced expression is related to cancer metastasis. The effect of IRBIT on NBCn1 and its relation to cancer cell migration remain obscure. We therefore aimed to determine the effect of IRBIT on NBCn1 and the regulation of cancer cell migration due to IRBIT-induced alterations in NBCn1 activity. Overexpression of IRBIT enhanced cancer cell migration and NBC activity. Knockdown of IRBIT or NBCn1 and treatment with an NBC-specific inhibitor, S0859, attenuated cell migration. Stimulation with oncogenic epidermal growth factor enhanced the expression of NBCn1 and migration of cancer cells by recruiting IRBIT. The recruited IRBIT stably maintained the expression of the NBCn1 transporter machinery in the plasma membrane. Combined inhibition of IRBIT and NBCn1 dramatically inhibited the migration of cancer cells. Combined modulation of IRBIT and NBCn1 offers an effective strategy for attenuating cancer metastasis.

7.
J Control Release ; 317: 181-194, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31785303

RESUMO

In the present study, we examined the potential of cell-penetrating peptide (CPP)-based intranasal drug delivery for the treatment of localized nasal diseases. Many charged or non-hydrophobic drugs have difficulty penetrating into the nasal epithelium due to intrinsic membrane impermeability and rapid mucociliary clearance in the nasal cavity. To treat chronic rhinosinusitis with nasal polyps (CRSwNP), one of the most common localized nasal diseases, we conjugated resveratrol (RSV) to an amphiphilic α-helical leucine (L)- and lysine (K)-rich CPP (LK) and intranasally delivered it to the interior of nasal epithelial cells for inhibiting epithelial-to-mesenchymal transition (EMT) caused by hypoxia-inducible factor 1α. The RSV-LK conjugate could penetrate into the nasal epithelium and efficiently inhibit EMT, nasal polyp formation, epithelial disruption, and related inflammation in an eosinophilic CRSwNP mouse model, at 10-fold lower doses and with 3-fold less frequent administration than free RSV. Due to the rapid penetration into the nasal epithelium and the therapeutic effect of the RSV-LK conjugate at much lower doses than free RSV, this CPP-based delivery system, with the ability to overcome the tight nasal epithelial barrier, may provide a new strategy for the treatment of localized nasal diseases without the systemic side effects of cargo drugs.


Assuntos
Peptídeos Penetradores de Células , Pólipos Nasais , Sinusite , Animais , Doença Crônica , Transição Epitelial-Mesenquimal , Camundongos , Mucosa Nasal , Pólipos Nasais/patologia , Resveratrol , Sinusite/tratamento farmacológico , Sinusite/patologia
9.
Molecules ; 24(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546841

RESUMO

Disulfiram has been used in the treatment of alcoholism and exhibits an anti-tumor effect. However, the intracellular mechanism of anti-tumor activity of Disulfiram remains unclear. In this study, we focused on the modulatory role of Disulfiram via oncogenic factor carbonic anhydrase CA12 and its associated transporter anion exchanger AE2 in lung cancer cell line A549. The surface expression of CA12 and AE2 were decreased by Disulfiram treatment with a time-dependent manner. Disulfiram treatment did not alter the expression of Na+-bicarbonate cotransporters, nor did it affect autophagy regulation. The chloride bicarbonate exchanger activity of A549 cells was reduced by Disulfiram treatment in a time-dependent manner without change in the resting pH level. The expression and activity of AE2 and the expression of CA12 were also reduced by Disulfiram treatment in the breast cancer cell line. An invasion assay and cell migration assay revealed that Disulfiram attenuated the invasion and migration of A549 cells. In conclusion, the attenuation of AE2 and its supportive enzyme CA12, and the inhibitory effect on cell migration by Disulfiram treatment in cancer cells provided the molecular evidence supporting the potential of Disulfiram as an anticancer agent.


Assuntos
Antineoplásicos/farmacologia , Anidrases Carbônicas/metabolismo , Movimento Celular/efeitos dos fármacos , Antiportadores de Cloreto-Bicarbonato/metabolismo , Dissulfiram/farmacologia , Reposicionamento de Medicamentos , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Invasividade Neoplásica , Microambiente Tumoral/efeitos dos fármacos
10.
J Enzyme Inhib Med Chem ; 34(1): 179-188, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30451023

RESUMO

Patients carrying the carbonic anhydrase12 E143K mutation showed the dry mouth phenotype. The mechanism underlying the modulation of aquaporin 5 and function in the salivary glands by carbonic anhydrase12 remains unknown. In this study, we identified the mislocalised aquaporin 5 in the salivary glands carrying the E143K. The intracellular pH of E143K cells was more acidic than that of the cells carrying wild type. To evaluate the role of carbonic anhydrase12 on the volume regulation of aquaporin 5, the submandibular gland cells were subjected to hypotonic stimuli. E143K enhanced the extent of swelling of cells on hypotonicity. Aquaporin 5 modulates water influx through ion transporters to prevent osmotic imbalance. These results suggest that the carbonic anhydrase12 E143K, including acidification or inflammation, mediates volume dysregulation by the loss of aquaporin 5. Thus, carbonic anhydrase12 may determine sensible effects on the cellular osmotic regulation by modulating aquaporin 5.


Assuntos
Aquaporina 5/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Mutação , Glândula Submandibular/enzimologia , Glândula Submandibular/metabolismo , Animais , Membrana Celular/enzimologia , Tamanho Celular , Estabilidade Enzimática , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Concentração Osmolar , Glândula Submandibular/citologia
11.
Mol Carcinog ; 58(5): 654-665, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30575123

RESUMO

Intermittent hypoxia (IH), a characteristic of obstructive sleep apnea, is known to promote cancer progression and aggressiveness in mouse models. However, little is known regarding the effect of IH on cancer initiation. Here, the effect of IH on carcinogenesis was explored in azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colon cancer models with three different protocols. In the first protocol, two other application time points (early or late initiation of IH) were applied. In the second protocol, mice were divided into only two groups, and then exposed to either N or IH conditions for 14 days. In the third protocol, a pharmacological inhibition study for anti-inflammation (5-aminosalicylate) or anti-oxidative stress (N-acetylcysteine [NAC]) was performed. The number of tumors was significantly higher in the IH-1 than in the N or IH-2 groups. 8-oxo-2'-deoxyguanosine (8-OHdG) levels were higher in tumors of the IH-1 group than in that of the N and IH-2 groups. Gene expression related to reactive oxygen species production was higher in the IH-1 group than in the N and IH-2 groups, and it showed a positive correlation with 8-OHdG levels. Prior to cancer development 8-OHdG levels were already elevated in colonic epithelial regions in the IH group, possibly due to an imbalance between oxidative stress and antioxidant systems. NAC treatment resulted in a significant reduction in the number of tumors in mice exposed to IH. In conclusion, IH promotes carcinogenesis in a chemically-induced colon cancer model where elevated 8-OHdG may contribute to the increased tumor induction.


Assuntos
Azoximetano/toxicidade , Carcinogênese/patologia , Colite/patologia , Neoplasias do Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Hipóxia/fisiopatologia , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Carcinógenos/toxicidade , Colite/induzido quimicamente , Colite/metabolismo , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos
12.
IEEE Trans Ultrason Ferroelectr Freq Control ; 63(10): 1505-1513, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27392348

RESUMO

In this paper, we investigated the mechanoresponse of C2C12 mesenchymal precursor cells to focused low-intensity pulsed ultrasound (FLIPUS). The setup has been developed for in vitro stimulation of adherent cells in the defocused far field of the ultrasound propagating through the bottom of the well plate. Twenty-four-well tissue culture plates, carrying the cell monolayers, were incubated in a temperature-controlled water tank. The ultrasound was applied at 3.6-MHz frequency, pulsed at 100-Hz repetition frequency with a 27.8% duty cycle, and calibrated at an output intensity of ISATA = 44.5 ±7.1 mW/cm2. Numerical sound propagation simulations showed no generation of standing waves in the well plate. The response of murine C2C12 cells to FLIPUS was evaluated by measuring activation of mechanosensitive transcription factors, i.e., activator protein-1 (AP-1), specificity protein 1 (Sp1), and transcriptional enhancer factor (TEAD), and expression of mechanosensitive genes, i.e., c-fos, c-jun, heparin binding growth associated molecule (HB-GAM), and Cyr-61. FLIPUS induced 50% ( p ≤ 0.05 ) and 70% ( p ≤ 0.05 ) increases in AP-1 and TEAD promoter activities, respectively, when stimulated for 5 min. The Sp1 activity was enhanced by about 20% ( p ≤ 0.05 ) after 5-min FLIPUS exposure and the trend persisted for 30-min ( p ≤ 0.05 ) and 1-h ( p ≤ 0.05 ) stimulation times. Expressions of mechanosensitive genes c-fos ( p ≤ 0.05 ), c-jun ( p ≤ 0.05 ), HB-GAM ( p ≤ 0.05 ), and cystein-rich protein 61 ( p ≤ 0.05 ) were enhanced in response to 5-min FLIPUS stimulation. The increase in proliferation of C2C12s occurred after the FLIPUS stimulation ( p ≤ 0.05 ), with AP-1, Sp1, and TEAD possibly regulating the observed cellular activities.


Assuntos
Mecanotransdução Celular , Células-Tronco Mesenquimais/fisiologia , Fatores de Transcrição/fisiologia , Ondas Ultrassônicas , Animais , Linhagem Celular , Camundongos
13.
Cell Rep ; 16(1): 28-36, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27320923

RESUMO

The Wnt/ß-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of ß-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic ß-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/ß-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/ß-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in vitro and in mouse cancer models. MSAB binds to ß-catenin, promoting its degradation, and specifically downregulates Wnt/ß-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/ß-catenin signaling pathway.


Assuntos
Benzoatos/farmacologia , Oncogenes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , meta-Aminobenzoatos/farmacologia , Animais , Benzoatos/química , Linhagem Celular Tumoral , Camundongos , Sulfonamidas/química , Ensaios Antitumorais Modelo de Xenoenxerto , meta-Aminobenzoatos/química
14.
Chem Biol ; 22(9): 1206-16, 2015 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-26320861

RESUMO

TP53 is the most frequently mutated gene in human cancer, and small-molecule reactivation of mutant p53 function represents an important anticancer strategy. A cell-based, high-throughput small-molecule screen identified chetomin (CTM) as a mutant p53 R175H reactivator. CTM enabled p53 to transactivate target genes, restored MDM2 negative regulation, and selectively inhibited the growth of cancer cells harboring mutant p53 R175H in vitro and in vivo. We found that CTM binds to Hsp40 and increases the binding capacity of Hsp40 to the p53 R175H mutant protein, causing a potential conformational change to a wild-type-like p53. Thus, CTM acts as a specific reactivator of the p53 R175H mutant form through Hsp40. These results provide new insights into the mechanism of reactivation of this specific p53 mutant.


Assuntos
Antineoplásicos/farmacologia , Dissulfetos/farmacologia , Proteínas de Choque Térmico HSP40/metabolismo , Alcaloides Indólicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Dissulfetos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Ensaios de Triagem em Larga Escala , Humanos , Alcaloides Indólicos/química , Camundongos , Camundongos Nus , Mutação , Bibliotecas de Moléculas Pequenas/química , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Science ; 349(6247): 1261669, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26228159

RESUMO

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.


Assuntos
Apoptose/imunologia , Proteínas de Membrana/metabolismo , Fagocitose/imunologia , Fosfatidilserinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Antígenos B7 , Linhagem Celular Tumoral , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Transdução de Sinais
16.
Life Sci ; 110(2): 93-8, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-24968300

RESUMO

AIMS: The objective of this study is to investigate glucosamine (GlcN) as a transcriptional regulator of iNOS and other genes in association with the dynamic O-GlcNAcylation of RNA polymerase II (RNAPII). MAIN METHODS: The LPS- and/or GlcN-stimulated transcriptional activities of various Gal4-binding site/TATA-box-containing reporter constructs were measured. KEY FINDINGS: Basal transcriptional activities of nuclear factor-κB (NF-κB) and nitric oxide synthase (iNOS) reporter plasmids are inhibited by GlcN in RAW264.7 cells. Furthermore, GlcN suppressed whereas lipopolysaccharide (LPS) stimulated the basal activity of Gal4-binding site/TATA-box-containing reporter constructs. LPS reduced the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of RNAPII, but enhanced the binding of this enzyme to the iNOS promoter. In contrast, GlcN enhanced RNAPII O-GlcNAcylation, but inhibited iNOS promoter binding. Furthermore, the basal activities of reporter plasmids containing activator protein 1 (AP1), E2F, or cyclic AMP response element (CRE) binding sites were consistently inhibited by GlcN in a dose-dependent manner. However, GlcN did not inhibit the phorbol 12-myristate 13-acetate- (PMA-) or forskolin-induced transcriptional activities of AP1 and CRE. The transcriptional activity of transforming growth factor alpha (TGF-α) was slightly increased by both LPS and GlcN. SIGNIFICANCE: In conclusion, our data demonstrate that LPS activates, whereas GlcN suppresses, basal activities of transcription through the regulation of RNAPII O-GlcNAcylation and DNA binding.


Assuntos
DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucosamina/metabolismo , Lipopolissacarídeos/metabolismo , RNA Polimerase II/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Análise de Variância , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Fatores de Transcrição E2F/metabolismo , Galactosiltransferases , Glucosamina/genética , Glucosamina/fisiologia , Immunoblotting , Luciferases , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Elementos Reguladores de Transcrição/genética , Fator de Transcrição AP-1/metabolismo , Aglutininas do Germe de Trigo
17.
Cell Rep ; 5(2): 331-9, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-24139803

RESUMO

Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo
18.
Am J Physiol Cell Physiol ; 305(6): C601-8, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23824843

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single ß-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-κB and inducible nitric oxide synthase (iNOS). In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor mammalian Sin3A (mSin3A). Furthermore, mSin3A, histone deacetylase (HDAC)1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A, and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor trichostatin A (TSA) on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases, p300, and cAMP response element-binding protein-binding protein (CBP) enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-κB activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.


Assuntos
Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Repressoras/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Camundongos , N-Acetilglucosaminiltransferases/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Gênica
19.
Br J Pharmacol ; 169(7): 1551-60, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23646894

RESUMO

BACKGROUND AND PURPOSE: Previously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on LPS-induced inducible NOS (iNOS) through the inhibition of NF-κB activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation. EXPERIMENTAL APPROACH: BV2 cells were stimulated with LPS with or without GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interactions were examined by co-immunoprecipitation. KEY RESULTS: LPS stimulated the activation of c-Rel, increased the O-GlcNAcylation of c-Rel and enhanced the binding of c-Rel to the NF-κB site in the iNOS promoter; GlcN attenuated these effects of LPS. O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel was increased by LPS and reduced by GlcN. LPS increased the interaction of c-Rel with O-GlcNAc transferase (OGT) and p50/p105, and GlcN suppressed these interactions. Knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, but did not affect either the binding of c-Rel to the iNOS promoter or the transcriptional activity of c-Rel. CONCLUSIONS AND IMPLICATIONS: In BV2 microglial cells, the anti-inflammatory effect of GlcN is mediated by prevention of the prolonged activation of transcription factors, c-Rel and NF-κB. Further clarification of the mechanism by which GlcN exerts this effect will facilitate the development of pharmacological strategies for preventing excessive NO formation when targeting inflammatory diseases of the periphery or CNS.


Assuntos
Acilação/efeitos dos fármacos , Glucosamina/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Glucosamina/química , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas
20.
Nitric Oxide ; 31: 1-8, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23454593

RESUMO

Expression of inducible nitric oxide synthase (iNOS) protein by lipopolysaccharide (LPS) in BV2 microglia cells increased in a biphasic manner. Glucosamine (GlcN) selectively suppressed the late- but not early-stage iNOS response to LPS. Prolonged induction of iNOS expression by LPS was inhibited by cycloheximide, suggesting that de novo protein synthesis was required. Late-phase activation of nuclear factor-kappaB (NF-κB) activity required for sustained iNOS induction. Nuclear translocation and DNA binding of NF-κB, and Rel proteins expressions were inhibited by GlcN at later time points but not upon immediate early-stage activation by LPS. We show that GlcN selectively inhibits sustained iNOS induction by inhibiting Rel protein expression at both the mRNA and protein levels; such expression is required for prolonged iNOS induction by LPS. Our results provide mechanistic evidence that GlcN regulates inflammation, represented by iNOS. The implication of these results is that GlcN may be a potent transcriptional regulator of iNOS and other genes involved in the general inflammation process.


Assuntos
Glucosamina/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Oncogênicas v-rel/antagonistas & inibidores , Animais , Western Blotting , Linhagem Celular , Indução Enzimática/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/biossíntese , NF-kappa B/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Oncogênicas v-rel/biossíntese , Proteínas Oncogênicas v-rel/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos
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