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1.
J Pharmacol Sci ; 140(1): 102-105, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31103330

RESUMO

Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.


Assuntos
Sinalização do Cálcio/fisiologia , Endotelina-1/farmacologia , Receptor de Endotelina A/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Feminino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/efeitos dos fármacos
2.
J Immunol Res ; 2018: 6249085, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977930

RESUMO

Toll/IL-1R-domain-containing adaptor-inducing IFN-ß (TRIF) is an important adaptor for TLR3- and TLR4-mediated inflammatory signaling pathways. Recent studies have shown that TRIF plays a key role in vessel inflammation and atherosclerosis; however, the precise mechanisms are unclear. We investigated the mechanisms of the TRIF-regulated inflammatory response in RAW264.7 macrophages under oxidized low-density lipoprotein (ox-LDL) stimulation. Our data show that ox-LDL induces TRIF, miR-155, and BIC expression, activates the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways, and elevates the levels of IL-6 and TNF-α in RAW264.7 cells. Knockdown of TRIF using TRIF siRNA suppressed BIC, miR-155, IL-6, and TNF-α expression and inhibited the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways. Inhibition of ERK1/2 signaling also suppressed BIC and miR-155 expression. These findings suggest that TRIF plays an important role in regulating the ox-LDL-induced macrophage inflammatory response and that TRIF modulates the expression of BIC/miR-155 and the downstream SOCS1-STAT3-NF-κB signaling pathway via ERK1/2. Therefore, TRIF might be a novel therapeutic target for atherosclerosis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , MicroRNAs/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Inativação Gênica , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/fisiologia , Camundongos , MicroRNAs/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , NF-kappa B/metabolismo , Células RAW 264.7 , Precursores de RNA/metabolismo , RNA Interferente Pequeno , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo
3.
J Cell Biochem ; 119(1): 123-129, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28574608

RESUMO

The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of maturation promoting factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription factors in cumulus cells or cytostatic factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Fator Promotor de Maturação/metabolismo , Meiose , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Humanos , Mamíferos , Fator Promotor de Maturação/fisiologia , Prófase Meiótica I , Metáfase , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Oócitos/enzimologia
4.
J Dermatol Sci ; 89(3): 241-247, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29198699

RESUMO

BACKGROUND: Mechanical stress is an ubiquitous challenge of human cells with fundamental impact on cell physiology. Previous studies have shown that stretching promotes signalling cascades involved in proliferation and tissue enlargement. OBJECTIVE: The present study is dedicated to learn more about cellular structures contributing to perception and signal transmission of cell stretch. In particular, we hypothesized that desmosmal contacts and the adjacent keratin filament build an intercellular matrix providing information about the mechanical load. METHODS: Epidermal cells with different keratin equipment were seeded on flexible silicon dishes and stretched. As read out parameter the activation of PKB/Akt and p44/42 was monitored by Western blotting. Likewise desomosomal contacts were manipulated by depletion or addition of calcium. Moreover, desmoglein 3 and desmocollin 3 were blocked by either specific antibodies or siRNA. RESULTS: It was found that the omission of calcium from the medium, a necessary cofactor for desmosomal cadherins, inhibited stretch mediated activation of PKB/Akt and p44/42. The relevance of desmosomes in this context was further substantiated by experiments using a desmoglein 3 blocking antibody (AK23) and siRNA against desmocollin 3. Moreover, disruption of the keratin filament by sodium orthovanadate also abrogates PKB/Akt and p44/42 activation in response to stretch. Likewise, KEB-7 keratinocytes harbouring a mutation in the keratin 14 gene and genetically modified keratinocytes devoid of any keratin show an altered signalling after stretch indicating the relevance of the keratin filament in this context. CONCLUSION: Besides their important role in cell architecture our results identify desmosomes and keratins as mechanosensing structures.


Assuntos
Desmossomos/fisiologia , Queratinas/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Cálcio/fisiologia , Células Cultivadas , Desmogleína 3/fisiologia , Ativação Enzimática , Humanos , Estresse Mecânico
5.
J Agric Food Chem ; 65(39): 8674-8682, 2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28873302

RESUMO

Drought stress is one of the most destructive environmental factors that affect tomato plants adversely. Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress. In this study, SlMAPK3 was induced by drought stress, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to generate slmapk3 mutants. Two independent T1 transgenic lines and wild-type (WT) tomato plants were used for analysis of drought tolerance. Compared with WT plants, slmapk3 mutants exhibited more severe wilting symptom, higher hydrogen peroxide content, lower antioxidant enzymes activities, and suffered more membrane damage under drought stress. Furthermore, knockout of SlMAPK3 led to up- or down-regulated expressions of drought stress-responsive genes including SlLOX, SlGST, and SlDREB. The results suggest that SlMAPK3 is involved in drought response in tomato plants by protecting cell membranes from oxidative damage and modulating transcription of stress-related genes.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Antioxidantes/análise , Membrana Celular/fisiologia , Secas , Peróxido de Hidrogênio/análise , Lycopersicon esculentum/química , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Mutagênese , Estresse Oxidativo , Plantas Geneticamente Modificadas
6.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 42(4): 380-388, 2017 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-28490694

RESUMO

OBJECTIVE: To observe effect of acupuncture combined with hypothermia therapy on MAPK/ERK pathway and apoptosis related factorsin rats suffered cerebral ischemia reperfusion and to explore underlying mechanisms.
 Methods: Middle cerebral artery ischemia model were established.Ninety SD rats were randomly assigned into a blank group, a control group, a model group, an acupuncture group, a mild hypothermia group, and an acupuncture with hypothermia group. After 72 h treatment, nerve function defect scores were observed, and infarction area percent was detected by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining; expressions of Bcl-2 and Bax were examined by immunohistochemistry; apoptotic cells were detected by TUNEL assay; and expression levels of phospho-mitogen-activated protein kinase(p-MEK2) and phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2) in the rats' hippocampus ischemic side were determined by Western blot.
 Results: In the rats of the model group, the neural function defect scores, the infarction area percent, the expression level of Bax, and apoptotic cells increased, while the level of Bcl-2 decreased significantly. The level of p-MEK2 and p-ERK1/2 increased obviously compared with the blank and control groups (P<0.05 or P<0.01). After treatment with acupuncture and hypothermia, the neural function defect scores, infarction area percent, and the level of Bax, apoptotic cells and the levels of p-MEK2 and p-ERK1/2 were significantly decreased, while the level of Bcl-2 in the treatment group was significantly elevated (P<0.05 or P<0.01) compared with the model group. Compared with the acupuncture group or the hypothermia group, the neural function defect scores and the levels of p-MEK2 and p-ERK1/2 in the acupuncture combined with hypothermia group were significantly reduced (P<0.05 or P<0.01).
 Conclusion: Acupuncture and hypothermia therapy can improve cerebral function, and reduce the cerebral injury through down-regulation of Bax level, and up-regulation of Bcl-2 level, which is related to reducing the levels of p-MEK2 and p-ERK1/2. The therapeutic effects on cerebral ischemia reperfusion injury for combination of acupuncture with hypothermia are better than those with single application of acupuncture or hypothermia.


Assuntos
Terapia por Acupuntura , Hipotermia Induzida , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Apoptose/fisiologia , Infarto Encefálico/terapia , Lesões Encefálicas/terapia , Isquemia Encefálica/terapia , Regulação para Baixo , MAP Quinase Quinase 2/fisiologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/terapia , Regulação para Cima
7.
BMC Biochem ; 18(1): 6, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28511672

RESUMO

BACKGROUND: Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. RESULTS: Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CONCLUSION: CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.


Assuntos
Chlamydomonas reinhardtii/genética , Regulação da Expressão Gênica de Plantas , Ferro/deficiência , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Inativação Gênica/fisiologia , Genes de Plantas , Proteína Quinase 3 Ativada por Mitógeno/genética , RNA Mensageiro/análise , Transdução de Sinais , Estresse Fisiológico
8.
Gastroenterology ; 153(2): 521-535.e20, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28438610

RESUMO

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Esvaziamento Gástrico/fisiologia , Hiperglicemia/fisiopatologia , Células Intersticiais de Cajal/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fatores de Transcrição/fisiologia , Animais , Feminino , Humanos , Células Intersticiais de Cajal/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Receptores para Leptina/genética , Regulação para Cima
9.
PLoS One ; 12(2): e0172466, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28222174

RESUMO

Several recent studies have reported on the role of mitogen-activated protein kinase (MAPK3) in plant immune responses. However, little is known about how MAPK3 functions in tomato (Solanum lycopersicum L.) infected with tomato yellow leaf curl virus (TYLCV). There is also uncertainty about the connection between plant MAPK3 and the salicylic acid (SA) and jasmonic acid (JA) defense-signaling pathways. The results of this study indicated that SlMAPK3 participates in the antiviral response against TYLCV. Tomato seedlings were inoculated with TYLCV to investigate the possible roles of SlMAPK1, SlMAPK2, and SlMAPK3 against this virus. Inoculation with TYLCV strongly induced the expression and the activity of all three genes. Silencing of SlMAPK1, SlMAPK2, and SlMAPK3 reduced tolerance to TYLCV, increased leaf H2O2 concentrations, and attenuated expression of defense-related genes after TYLCV infection, especially in SlMAPK3-silenced plants. Exogenous SA and methyl jasmonic acid (MeJA) both significantly induced SlMAPK3 expression in tomato leaves. Over-expression of SlMAPK3 increased the transcript levels of SA/JA-mediated defense-related genes (PR1, PR1b/SlLapA, SlPI-I, and SlPI-II) and enhanced tolerance to TYLCV. After TYLCV inoculation, the leaves of SlMAPK3 over-expressed plants compared with wild type plants showed less H2O2 accumulation and greater superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activity. Overall, the results suggested that SlMAPK3 participates in the antiviral response of tomato to TYLCV, and that this process may be through either the SA or JA defense-signaling pathways.


Assuntos
Begomovirus/fisiologia , Ciclopentanos/farmacologia , Lycopersicon esculentum/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Oxilipinas/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/fisiologia , Ácido Salicílico/farmacologia , Transdução de Sinais/fisiologia , Resistência à Doença , Indução Enzimática , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Lycopersicon esculentum/virologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/genética , Estresse Oxidativo , Oxirredutases/biossíntese , Oxirredutases/genética , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/enzimologia , Folhas de Planta/virologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transdução de Sinais/genética , Transcrição Genética
10.
Korean J Parasitol ; 55(6): 613-622, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29320816

RESUMO

IL-12 and IL-23 are closely related in structure, and have been shown to play crucial roles in regulation of immune responses. However, little is known about the regulation of these cytokines in T cells. Here, we investigated the roles of PI3K and MAPK pathways in IL-12 and IL-23 production in human Jurkat T cells in response to Toxoplasma gondii and LPS. IL-12 and IL-23 production was significantly increased in T cells after stimulation with T. gondii or LPS. T. gondii and LPS increased the phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK1/2 in T cells from 10 min post-stimulation, and peaked at 30-60 min. Inhibition of the PI3K pathway reduced IL-12 and IL-23 production in T. gondii-infected cells, but increased in LPS-stimulated cells. IL-12 and IL-23 production was significantly reduced by ERK1/2 and p38 MAPK inhibitors in T. gondii- and LPS-stimulated cells, but not in cells treated with a JNK1/2 inhibitor. Collectively, IL-12 and IL-23 production was positively regulated by PI3K and JNK1/2 in T. gondii-infected Jurkat cells, but negatively regulated in LPS-stimulated cells. And ERK1/2 and p38 MAPK positively regulated IL-12 and IL-23 production in Jurkat T cells. These data indicate that T. gondii and LPS induced IL-12 and IL-23 production in Jurkat T cells through the regulation of the PI3K and MAPK pathways; however, the mechanism underlying the stimulation of IL-12 and IL-23 production by T. gondii in Jurkat T cells is different from that of LPS.


Assuntos
Interleucina-12/metabolismo , Interleucina-23/metabolismo , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Toxoplasma/imunologia , Células Cultivadas , Humanos , Células Jurkat , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
11.
J Leukoc Biol ; 99(2): 311-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26336156

RESUMO

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.


Assuntos
Granulócitos/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Animais , Butadienos/farmacologia , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Mielopoese/fisiologia , Nitrilos/farmacologia , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/fisiologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , RNA Interferente Pequeno/genética
12.
Food Funct ; 7(1): 84-92, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26645329

RESUMO

This paper investigated if marginal zinc nutrition during gestation could affect fetal exposure to glucocorticoids as a consequence of a deregulation of placental 11ßHSD2 expression. Placenta 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) plays a central role as a barrier protecting the fetus from the deleterious effects of excess maternal glucocorticoids. Rats were fed control (25 µg zinc per g diet) or marginal (10 µg zinc per g diet, MZD) zinc diets from day 0 through day 19 (GD19) of gestation. At GD19, corticosterone concentration in plasma, placenta, and amniotic fluid was similar in both groups. However, protein and mRNA levels of placenta 11ßHSD2 were significantly higher (25% and 58%, respectively) in MZD dams than in controls. The main signaling cascades modulating 11ßHSD2 expression were assessed. In MZD placentas the activation of ERK1/2 and of the downstream transcription factor Egr-1 was low, while p38 phosphorylation and SP-1-DNA binding were low compared to the controls. These results point to a central role of ERK1/Egr-1 in the regulation of 11ßHSD2 expression under the conditions of limited zinc availability. In summary, results show that an increase in placenta 11ßHSD2 expression occurs as a consequence of gestational marginal zinc nutrition. This seems to be due to a low tissue zinc-associated deregulation of ERK1/2 rather than to exposure to high maternal glucocorticoid exposure. The deleterious effects on brain development caused by diet-induced marginal zinc deficiency in rats do not seem to be due to fetal exposure to excess glucocorticoids.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Placenta/enzimologia , Zinco/deficiência , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/análise , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Animais , Dieta , Feminino , Expressão Gênica/fisiologia , Idade Gestacional , Glucocorticoides/análise , Masculino , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Placenta/química , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Zinco/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
14.
Adv Exp Med Biol ; 860: 269-77, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26303491

RESUMO

The carotid body is the main mammalian oxygen-sensing organ regulating ventilation. Despite the carotid body is subjected of extensive anatomical and functional studies, little is yet known about the molecular pathways signaling the neurotransmission and neuromodulation of the chemoreflex activity. As kinases are molecules widely involved in motioning a broad number of neural processes, here we hypothesized that pathways of protein kinase B (AKT) and extracellular signal-regulated kinases ½ (ERK1/2) are implicated in the carotid body response to hypoxia. This hypothesis was tested using the in-vitro carotid body/carotid sinus nerve preparation ("en bloc") from Sprague Dawley adult rats. Preparations were incubated for 60 min in tyrode perfusion solution (control) or containing 1 µM of LY294002 (AKT inhibitor), or 1 µM of UO-126 (ERK1/2 inhibitor). The carotid sinus nerve chemoreceptor discharge rate was recorded under baseline (perfusion solution bubbled with 5 % CO(2) balanced in O(2)) and hypoxic (perfusion solution bubbled with 5 % CO(2) balanced in N(2)) conditions. Compared to control, both inhibitors significantly decreased the normoxic and hypoxic carotid body chemoreceptor activity. LY294002- reduced carotid sinus nerve discharge rate in hypoxia by about 20 %, while UO-126 reduces the hypoxic response by 45 %. We concluded that both AKT and ERK1/2 pathways are crucial for the carotid body intracellular signaling process in response to hypoxia.


Assuntos
Corpo Carotídeo/fisiologia , Hipóxia/fisiopatologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Butadienos/farmacologia , Cromonas/farmacologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Nitrilos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley
15.
J Physiol ; 593(17): 3973-89, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26174421

RESUMO

Ghrelin, a hunger signalling peptide derived from the peripheral tissues, overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. Using in vivo and in vitro electrophysiological techniques, we show that ghrelin hyperpolarizes neurons and inhibits currents evoked by leptin and CCK-8. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition. The inhibitory actions of ghrelin were also abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K(+) conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK-8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin- or CCK-8-evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways.


Assuntos
Grelina/farmacologia , Canais KATP/fisiologia , Gânglio Nodoso/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Colecistocinina/farmacologia , Ingestão de Alimentos , Canais KATP/antagonistas & inibidores , Canais KATP/genética , Leptina/farmacologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Gânglio Nodoso/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos
16.
Bull Exp Biol Med ; 159(1): 58-61, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26033591

RESUMO

We studied the role of intracellular signaling molecules PI3K, МАРK ERK1/2, and р38 in stimulation of realization of the growth potential of mesenchymal progenitor cells by alkaloid songorine in vitro. Inhibitors of PI3K, ERK1/2 and р38 canceled the increase in proliferative activity of progenitor cells, the blockers of ERK1/2 and р38 reduced the intensity of progenitor cell differentiation.


Assuntos
Alcaloides/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Ensaio de Unidades Formadoras de Colônias , Flavonoides/farmacologia , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Camundongos , Camundongos Endogâmicos CBA , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Modelos Biológicos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
17.
Circ Res ; 117(3): 279-88, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26058828

RESUMO

RATIONALE: Reduction of myocardial infarct size by remote ischemic preconditioning (RIPC), that is, cycles of ischemia/reperfusion in an organ remote from the heart before sustained myocardial ischemia/reperfusion, was confirmed in all species so far, including humans. OBJECTIVE: To identify myocardial signal transduction of cardioprotection by RIPC. METHODS AND RESULTS: Anesthetized pigs were subjected to RIPC (4×5/5 minutes hindlimb ischemia/reperfusion) or placebo (PLA) before 60/180 minutes coronary occlusion/reperfusion. Phosphorylation of protein kinase B, extracellular signal-regulated kinase 1/2 (reperfusion injury salvage kinase [RISK] pathway), and signal transducer and activator of transcription 3 (survival activating factor enhancement [SAFE] pathway) in the area at risk was determined by Western blot. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Plasma sampled after RIPC or PLA, respectively, was transferred to isolated bioassay rat hearts subjected to 30/120 minutes global ischemia/reperfusion. RIPC reduced infarct size in pigs to 16±11% versus 43±11% in PLA (% area at risk; mean±SD; P<0.05). RIPC increased the phosphorylation of signal transducer and activator of transcription 3 at early reperfusion, and AG490 abolished the protection, whereas RISK blockade did not. Signal transducer and activator of transcription 5 phosphorylation was decreased at early reperfusion in both RIPC and PLA. In isolated rat hearts, pig plasma taken after RIPC reduced infarct size (25±5% of ventricular mass versus 38±5% in PLA; P<0.05) and activated both RISK and SAFE. RISK or SAFE blockade abrogated this protection. CONCLUSIONS: Cardioprotection by RIPC in pigs causally involves activation of signal transducer and activator of transcription 3 but not of RISK. Protection can be transferred with plasma from pigs to isolated rat hearts where activation of both RISK and SAFE is causally involved. The myocardial signal transduction of RIPC is the same as that of ischemic postconditioning.


Assuntos
Transfusão de Sangue , Membro Posterior/irrigação sanguínea , Precondicionamento Isquêmico/métodos , Infarto do Miocárdio/terapia , Proteínas Quinases/fisiologia , Ratos Endogâmicos Lew/fisiologia , Transdução de Sinais/fisiologia , Porco Miniatura/fisiologia , Animais , Pressão Sanguínea , Circulação Coronária , Hemodinâmica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Infarto do Miocárdio/sangue , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica , Especificidade de Órgãos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Fator de Transcrição STAT3/fisiologia , Fator de Transcrição STAT5/fisiologia , Transdução de Sinais/efeitos dos fármacos , Suínos , Porco Miniatura/sangue
18.
Exp Hematol ; 43(7): 524-33.e1, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25846811

RESUMO

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Adulto , Idoso , Antineoplásicos/farmacologia , Apoptose , Butadienos/farmacologia , Linhagem Celular Tumoral , Interações de Medicamentos , Fosfatase 6 de Especificidade Dupla/fisiologia , Ativação Enzimática , Retroalimentação Fisiológica , Feminino , Glucose/farmacologia , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mielomonocítica Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metformina/farmacologia , Pessoa de Meia-Idade , Nitrilos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas
19.
Arthritis Rheumatol ; 67(7): 1760-5, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25833292

RESUMO

OBJECTIVE: Dysregulated apoptosis of monocytes is a pathogenic feature of rheumatoid arthritis (RA). The aim of this study was to investigate the role of TRAIL and TRAIL-induced apoptosis in patients with RA. METHODS: Cell surface expression and serum concentrations of TRAIL were determined in 63 patients with RA, and TRAIL-induced monocyte apoptosis was quantified. Surface expression of TRAILR-1, TRAILR-2, TRAILR-3, TRAILR-4, CXCR1, and CXCR2 was determined, and intracellular signal transduction was investigated. In 8 patients with RA, clinical and laboratory parameters of disease activity were investigated longitudinally, before and after initiation of treatment with tumor necrosis factor (TNF) inhibitors. RESULTS: Serum concentrations of both TRAIL and interleukin-8 (IL-8) were increased in patients with RA, while cell surface expression of the TRAIL receptors TRAILR-1, TRAILR-2, TRAILR-3, and TRAILR-4 was diminished. TRAIL-induced monocyte apoptosis was significantly decreased in RA due to increased TRAIL-induced IL-8 secretion by RA monocytes. The combined effect of TRAIL and IL-8 on monocytes resulted in activation of antiapoptotic pathways, including p42/44 MAPK and p38. Susceptibility to TRAIL-induced apoptosis was restored in RA monocytes after 3 months of TNF inhibition. CONCLUSION: In RA, circulating monocytes with the potential to produce proinflammatory cytokines appear to have defects in several pathways of apoptosis induction, among which is a deficiency in TRAIL-induced apoptosis. Although this resistance to apoptosis might contribute to perpetuation of the disease, it remains to be determined whether specific induction of apoptosis could be therapeutically beneficial.


Assuntos
Apoptose/efeitos dos fármacos , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Comunicação Autócrina/fisiologia , Monócitos/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/fisiologia , Estudos de Casos e Controles , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Monócitos/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
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