RESUMO
Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.
Assuntos
Membrana Celular/metabolismo , Lisossomos/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Western Blotting , Endotelina-1/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Mutação , Fosforilação , Transporte Proteico/efeitos dos fármacos , Receptor de Endotelina A/agonistas , Receptor de Endotelina A/genética , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Ubiquitinação/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤ 15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥ 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.
Assuntos
Citotoxinas/isolamento & purificação , Filtração/normas , Gases/química , Fumaça/análise , Produtos do Tabaco/análise , Animais , Soluções Tampão , Linhagem Celular Tumoral , Citotoxinas/toxicidade , Vidro/química , Humanos , Hidrocarbonetos/isolamento & purificação , Fosfatos/química , Ratos , Padrões de Referência , Reprodutibilidade dos Testes , Fumaça/efeitos adversos , Temperatura , Fatores de TempoRESUMO
AIMS: Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitivity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR-mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat skeletal muscle. MAIN METHODS: Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) was overexpressed in L6 myoblasts using adenovirus-mediated gene transfer. KEY FINDINGS: ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was abolished by BQ123 (a selective ET type A receptor (ETAR) antagonist), YM-254890 (a Gαq/11 protein inhibitor), and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholipase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of dominant-negative dynamin (K44A). These results suggest that ETAR stimulation induces ERK1/2 phosphorylation in L6 myoblasts through Gq/11 protein-dependent, PLC-independent PDGFR transactivation which requires dynamin-dependent ETAR internalization. SIGNIFICANCE: Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the development of insulin sensitivity, the ETAR-mediated PDGFR transactivation and subsequent ERK1/2 activation play an important role in ET-1-induced insulin resistance.
Assuntos
Endotelina-1/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mioblastos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Ativação Transcricional , Animais , Cálcio/metabolismo , Linhagem Celular , Dinaminas/genética , Regulação Enzimológica da Expressão Gênica , Técnicas de Transferência de Genes , Genes Dominantes , Insulina/metabolismo , Resistência à Insulina , Músculo Esquelético/metabolismo , Fosforilação , Ratos , Receptor de Endotelina A/metabolismo , Transdução de SinaisRESUMO
Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0µM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent cytotoxicity with an EC50 value of 264.0±16.9µM (n=3). The concentrations of acrolein, MVK and CPO in the CSE were 3368±334, 2429±123 and 392.9±31.8µM (n=4), respectively, which were higher than the cytotoxic concentrations. The cytotoxicity of acrolein and MVK consisted of plasma membrane damage and decreased cell viability: the plasma membrane damage was totally prevented by treatment with an inhibitor of PKC or NOX, whereas the decreased cell viability was only partially prevented by these inhibitors. The cytotoxicity of CPO consisted only of decreased cell viability, which was totally resistant to these inhibitors. These results show that acrolein and MVK are responsible for the acute cytotoxicity of the CSE through PKC/NOX-dependent and -independent mechanisms, whereas CPO is responsible for the delayed cytotoxicity of the CSE through a PKC/NOX-independent mechanism.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Poluição por Fumaça de Tabaco/análise , Butanonas/química , Butanonas/toxicidade , Linhagem Celular Tumoral , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Ciclopentanos/química , Ciclopentanos/toxicidade , Interpretação Estatística de Dados , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidroxilaminas , Indicadores e Reagentes , Espectrometria de Massas , NADPH Oxidases/antagonistas & inibidores , Nicotina/química , Proteína Quinase C/antagonistas & inibidores , Alcatrões/farmacologiaRESUMO
Nicotine- and tar-free cigarette smoke extract (CSE) is reported to induce cell damage via activation of protein kinase C (PKC) and NADPH oxidase (NOX) in rat C6 glioma cells. Here we determined PKC isozyme(s) activated by CSE and their activation mechanism. In C6 glioma cells, mRNAs for PKCα, PKCδ, PKCε, and PKCι were expressed. CSE triggered translocation of PKCα and PKCε to plasma membrane. CSE-induced cell damage and PKC translocation were inhibited by chelating intracellular Ca(2+) but not extracellular Ca(2+). These results suggest that CSE induces cell damage through intracellular Ca(2+)-dependent activation of PKCα and PKCε and subsequent NOX activation.
Assuntos
Glioma/enzimologia , Glioma/patologia , Nicotina/efeitos adversos , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Animais , Cálcio/metabolismo , Cálcio/fisiologia , Membrana Celular/enzimologia , Ativação Enzimática , NADPH Oxidases/metabolismo , Ratos , Espécies Reativas de Oxigênio , Alcatrões , Células Tumorais CultivadasRESUMO
Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum (ER) Ca(2+) sensor to control ER Ca(2+) levels. A recent study has shown that STIM1L, a new splice variant of STIM1, is expressed in various tissues of rodent and in human skeletal muscle, and that the interaction of STIM1L with actin filament allows rapid activation of store-operated Ca(2+) entry (SOCE) mediated through Orai1 channels. Here, we characterize mRNA expression and function of human STIM1 and STIM1L, and compare their binding property to Orai1 functioning as store-operated Ca(2+) channels (SOCCs), and TRPC3 (transient receptor potential canonical 3) and TRPC6 channels functioning as endothelin type A receptor (ET(A)R)-operated Ca(2+) channels (ROCCs). Although mRNA for STIM1 was ubiquitously expressed in human tissues, STIM1L was detected only in skeletal muscle. STIM1L augmented thapsigargin- and endothelin-1-induced SOCE more strongly than STIM1 in human embryonic kidney 293 cells stably expressing ET(A)R, whereas, it tends to suppress ET(A)R-operated Ca(2+) entry (ROCE) via TRPC3 and TRPC6 more strongly than STIM1. Coimmunoprecipitation experiments have revealed that when compared with STIM1, STIM1L binds more abundantly to Orai1 and also to TRPC3 and TRPC6. These results suggest that the higher binding capacity of STIM1L to SOCCs and ROCCs plays an important role in the regulation of Ca(2+) signaling such as the augmentation of SOCE via Orai1 and the inhibition of ROCE via TRPC3 and TRPC6.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cátion TRPC/metabolismo , Cálcio/metabolismo , Ventrículos Cerebrais/metabolismo , Feminino , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas de Membrana/genética , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1 , Placenta/metabolismo , Gravidez , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Molécula 1 de Interação Estromal , Canal de Cátion TRPC6RESUMO
In this study, we report the unique role of arachidonate 5-lipoxygenase (Alox5) in the regulation of specific humoral immune responses. We previously reported an L22 monoclonal antibody with which human primary resting B cells in the mantle zones of lymphoid follicles are well-defined. Proteomics analyses enabled identification of an L22 antigen as Alox5, which was highly expressed by naive and memory B cells surrounding germinal centers. Cellular growth of mantle cell lymphoma cells also seemed to depend on Alox5. Alox5(-/-) mice exhibited weak antibody responses specific to foreign antigens at the initial and recall phases. This was probably attributable to the low number of follicular and memory B cells and the functional loss of interleukin-21-mediated responses of follicular B cells. Moreover, Alox5(-/-) mice could not fully foster the development of follicular B helper T (Tfh) cells even after immunization with foreign antigens. Further experiments indicated that Alox5 affected mortality in experimentally induced enterocolitis in germ-prone circumstances, indicating that Alox5 would endow immunologic milieu. Our results illustrate the novel role of Alox5 in adaptive humoral immunity by managing primary B cells and Tfh cells in vivo.