RESUMO
Extracellular matrix mineralization is critical for osteogenesis, and its dysregulation could result in osteoporosis and vascular calcification. IKK/NF-κB activation inhibits differentiation of osteoblasts, and reduces extracellular matrix mineralization, however the underlying mechanisms are poorly understood. In this study, we used CRISPR/Cas9 system to permanently inactivate IKKß in preosteoblast cells and confirmed that such cells displayed dramatic increase in extracellular matrix mineralization associated with JNK phosphorylation. Such observation was also found in our study using IKKß-deficient primary murine osteoblasts. Interestingly, we found that in Ikbkb-/-Mapk8-/- or Ikbkb-/-Mapk9-/- double knockout cells, the enhanced mineralization caused by IKKß deficiency was completely abolished, and deletion of either Mapk8 or Mapk9 was sufficient to dampen c-Jun phosphorylation. In further experiments, we discovered that absence of JNK1 or JNK2 on IKKß-deficient background resulted in highly conserved transcriptomic alteration in response to osteogenic induction. Therefore, identification of the indispensable roles of JNK1 and JNK2 in activating c-Jun and promoting osteoblast differentiation on IKKß-deficient background provided novel insights into restoring homeostasis in extracellular matrix mineralization.
Assuntos
Calcificação Fisiológica/genética , Matriz Extracelular/metabolismo , Quinase I-kappa B/genética , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Osteoblastos/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Células Cultivadas , Matriz Extracelular/genética , Técnicas de Inativação de Genes , Quinase I-kappa B/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Osteoblastos/fisiologia , Osteogênese/genética , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.
Assuntos
Anel Fibroso/patologia , Vértebras Cervicais/patologia , Disco Intervertebral/patologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Escoliose/etiologia , Fusão Vertebral , Animais , Anel Fibroso/enzimologia , Diferenciação Celular , Proliferação de Células , Vértebras Cervicais/enzimologia , Condrogênese , Feminino , Disco Intervertebral/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosforilação , Escoliose/enzimologia , Escoliose/patologiaRESUMO
AIM: The airway epithelium of smokers exhibits upregulated SPRR3, an indicator of pathogenic keratinization. The mechanisms underlying this phenomenon require investigation. PATIENTS & METHODS: Human bronchial epithelial (HBE) SPRR3 expression was analyzed by smoking status. Primary HBE cells were exposed to cigarette smoke (CS). SPRR3 expression, SPRR3 promoter activity, AP-1 factor binding and AP-1 factors' effects were analyzed. RESULTS: Current smokers display SPRR3 upregulation relative to never smokers. CS upregulates SPRR3 transcription in an exposure-dependent manner. CS promotes c-Jun and Fra1 binding to the SPRR3-AP-1/TRE site. Wild-type c-Jun and Fra1 upregulate, whereas c-Jun and Fra1, dominant-negative mutants, suppress SPRR3 promoter activity. CONCLUSION: CS induces SPRR3 upregulation in HBE cells by promoting aberrant c-Jun/Fra1 dimerization.
Assuntos
Brônquios/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/genética , Multimerização Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-jun/química , Fumar Tabaco/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Regiões Promotoras Genéticas , Fator de Transcrição AP-1/fisiologia , Regulação para CimaRESUMO
PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide, and its outcome is poor. The purpose of this study was to determine the association between JNK1 and vitamin D receptor (VDR) expression and the prognosis of ESCC. METHODS: Immunohistochemical staining was conducted on ESCC tissue microarrays (362 pairs of ESCC and normal esophagus tissues). The epithelial and stromal expression levels of c-jun NH2-terminal kinase 1 (JNK1) and VDR were scored and correlated with the ESCC characteristics. Laser-capture-based quantitative RT-PCR was performed on ESCC tissues. The effects of JNK1 and VDR on ESCC cell proliferation and migration were analyzed in vitro by transient transfection, and protein changes were evaluated by immunoblotting. RESULTS: Both JNK1 and VDR were reduced in ESCC epithelial cells in comparison with the normal esophagus, but the expression of JNK1 and VDR in ESCC stromal tissues, not epithelial cells, was strongly associated with the survival time of ESCC patients. Functional studies showed that increased JNK1 suppressed cancer cell proliferation, mobility, and migration, which were linked to the alterations of VDR and metastasis-associated proteins. CONCLUSION: JNK1 and VDR act as tumor suppressors, and their stromal expression levels are associated with prognosis in esophageal squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas/mortalidade , Neoplasias Esofágicas/mortalidade , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Receptores de Calcitriol/fisiologia , Adulto , Idoso , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/química , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Esôfago/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/análise , Prognóstico , Receptores de Calcitriol/análise , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The hypoxic response is a stress response triggered by low oxygen tension. Hypoxia-inducible factors (HIFs) play a prominent role in the pathobiology of hypoxia-associated conditions, including pulmonary hypertension (PH) and polycythemia. The c-Jun N-terminal protein kinase (JNK), a stress-activated protein kinase that consists of two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3, has been shown to be activated by hypoxia. However, the physiological role of JNK1 and JNK2 in the hypoxic response remains elusive. Here, using genetic knockout cells and/or mice, we show that JNK2, but not JNK1, up-regulates the expression of HIF-1α and HIF-2α and contributes to hypoxia-induced PH and polycythemia. Knockout or silencing of JNK2, but not JNK1, prevented the accumulation of HIF-1α in hypoxia-treated cells. Loss of JNK2 resulted in a decrease in HIF-1α and HIF-2α mRNA levels under resting conditions and in response to hypoxia. Consequently, hypoxia-treated Jnk2-/- mice had reduced erythropoiesis and were less prone to polycythemia because of decreased expression of the HIF target gene erythropoietin (Epo). Jnk2-/- mice were also protected from hypoxia-induced PH, as indicated by lower right ventricular systolic pressure, a process that depends on HIF. Taken together, our results suggest that JNK2 is a positive regulator of HIFs and therefore may contribute to HIF-dependent pathologies.
Assuntos
Hipóxia Celular/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Eritropoese/fisiologia , Eritropoetina/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Policitemia/metabolismo , RNA Mensageiro/genética , Ativação Transcricional , Regulação para CimaRESUMO
PURPOSE OF REVIEW: This review aims to assess the epidemiological evidence for a link between type 2 diabetes and hepatocellular carcinoma and to investigate possible pathophysiological mechanisms. RECENT FINDINGS: The presence of type 2 diabetes significantly increases the risk of developing hepatocellular carcinoma, and treatment with metformin may be associated with a lower risk. Treatment with insulin and sulphonylureas may be associated with increased risk. The pathophysiology underlying development of hepatocellular carcinoma in this context is complex and is likely to involve increased proinflammatory mediators, oxidative stress, JNK-1 activation, increased IGF-1 activity, altered gut microbiota and immunomodulation. Hepatocellular carcinoma incidence is increasing and this is likely to be linked to the increasing incidence of type 2 diabetes, obesity and the metabolic syndrome. These conditions increase the risk of developing hepatocellular carcinoma, and a greater understanding of the underlying pathophysiology may help with the development of novel treatments.
Assuntos
Carcinoma Hepatocelular/etiologia , Diabetes Mellitus Tipo 2/complicações , Neoplasias Hepáticas/etiologia , Animais , Humanos , Síndrome Metabólica/complicações , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Obesidade/complicações , Fatores de RiscoRESUMO
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/fisiologiaRESUMO
Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory.
Assuntos
Medo/efeitos dos fármacos , Medo/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Animais , Condicionamento Clássico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genéticaAssuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Fator de Transcrição Sp1/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína 1 Parceira de Translocação de RUNX1RESUMO
The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Produtos do Gene tax/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteínas de Neoplasias/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular , Sequência Consenso , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Humanos , Células Jurkat , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/fisiologia , Transfecção , Tretinoína/farmacologiaRESUMO
Increased training often results in stronger memories but the neural changes responsible for these stronger memories are poorly understood. It is proposed here that higher levels of training that result in stronger memories recruit additional cell signaling cascades. This study specifically examined if c-Jun N-terminal kinase 1 (JNK1) is involved in the formation of stronger fear conditioning memories. Wildtype (WT), JNK1 heterozygous (Het), and JNK1 knockout (KO) mice were fear conditioned with 1 trial, 2 trials, or 4 trials. All mice learned both contextual (hippocampus-dependent) and cued (hippocampus-independent) fear conditioning but for contextual fear conditioning only, the JNK1 KO mice did not show higher levels of learning with increased trials. That is, WT mice showed a significant linear increase in contextual fear conditioning as training trials increased from 1 to 2 to 4 trials whereas KO mice showed the same level of contextual fear conditioning as WT mice for 1 trial training but did not have increased levels of contextual fear conditioning with additional trials. These data suggest that JNK1 may not be critical for learning but when higher levels of hippocampus-dependent learning occur, JNK1 signaling is recruited and is necessary for stronger hippocampus-dependent memory formation.
Assuntos
Condicionamento Clássico/fisiologia , Medo/fisiologia , Aprendizagem/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Transdução de Sinais , Animais , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genética , Atividade MotoraRESUMO
Liver regeneration is controlled by a complex network of signaling molecules, and a prominent role for c-jun N-terminal kinase has been suggested during this process. In the present study, we aimed to characterize and define the cell-type-specific contribution of JNK1 activation during liver regeneration. We used hepatocyte-specific JNK1 knockout mice (JNK1(Δhepa)) using the cre/lox-P system. We performed partial hepatectomy (PH) in WT, JNK1(Δhepa) and JNK1(-/-) animals and investigated time-points up to 72 h after PH. Additionally, bone marrow transplantation experiments were conducted in order to identify the contribution of hematopoietic cell-derived JNK1 activation for liver regeneration. Our results show that liver regeneration was significantly impaired in JNK1(-/-) compared to JNK1(Δhepa) and WT animals. These data were evidenced by lower BrdU incorporation and decreased cell cycle markers such as Cyclin A, Cyclin D, E2F1 and PCNA 48 h after PH in JNK1(-/-) compared with JNK1(Δhepa) and WT livers. In JNK1(-/-) mice, our findings were associated with a reduced acute phase response as evidenced by a lower activation of the IL-6/STAT3/SAA-1 cascade. Additionally, CD11b(+)Ly6G(+)-cells were decreased in JNK1(-/-) compared with JNK1(Δhepa) and WT animals after PH. The transplantation of bone marrow-derived JNK1(-/-) into WT recipients caused significant reduction in liver regeneration. Interestingly, the transplantation of JNK1(-/-) into mice lacking JNK1 in hepatocytes only partially delayed liver regeneration. In summary, we provide evidence that (1) JNK1 in hematopoietic cells is crucial for liver regeneration, and (2) a synergistic function between JNK1 in hepatocytes and hematopoietic-derived cells is involved in the hepatic regenerative response.
Assuntos
Medula Óssea/enzimologia , Regeneração Hepática/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Insulin resistance is often associated with obesity and is a major risk factor for development of type 2 diabetes as well as cardiovascular and hepatic diseases. Insulin resistance may also increase the incidence or the aggressiveness of some cancers. Insulin resistance occurs owing to defects in insulin signaling in target tissues of this hormone. During the last ten years, it became evident that the chronic low-grade inflammatory state that develops during obesity plays an important role in insulin resistance development. Indeed, inflammatory cytokines activate several signaling pathways that impinge on the insulin signaling pathway. Among them, this review will focus on the implication of the MAP kinases JNK and ERK1/2 signaling in the development of insulin signaling alterations and will discuss the possibility to target these pathways in order to fight insulin resistance.
Assuntos
Inflamação/enzimologia , Resistência à Insulina , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Obesidade/imunologia , Obesidade/metabolismo , Animais , Humanos , Inflamação/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development. METHODS: Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing. RESULTS: By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs. CONCLUSIONS: JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.
Assuntos
Elasticidade/fisiologia , Fibroblastos/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Alvéolos Pulmonares/enzimologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Ativação Enzimática/fisiologia , Pulmão/citologia , Pulmão/enzimologia , Pulmão/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Alvéolos Pulmonares/citologiaRESUMO
Our previous results have indicated that Akt mediates 17ß-estradiol (E2) and/or estrogen receptor α (ERα) to inhibit lipopolysaccharide (LPS)-induced JNK activity, tumor necrosis factor α (TNFα) protein expression, and exhibits cardioprotective effects. Toll-like receptor 4 (TLR4) mRNAs often contain AU-rich elements (AREs) in their 3'-untranslated regions (3'UTR) which have a high affinity for RNA-binding proteins. It is not known whether E2 and ERα affect TLR4 mRNA stability and TLR4 protein expression through regulating the RNA-binding proteins, human antigen R (HuR), tristetraprolin (TTP) and AU-binding factor 1 (AUF-1) in myocardial cells. Therefore, we investigated if the LPS in- duces these RNA-binding proteins to regulate TLR4 mRNAs of cardiomyocytes, and whether the E2/ERα reduces the TLR4 mRNA stability induced by LPS through the inhibition of RNA-binding protein expression. Using a doxycycline (Dox)-induced Tet-On ERα H9c2 myocardic cell model, we also aimed to identify whether E2 and/or ERα regulate LPS-induced TLR4 mRNA stability. The results of Western blotting and reverse transcription-PCR assays demonstrated that LPS significantly in- creased the level of cytoplasmic HuR protein and the stability of TLR4 mRNA, and farther induced TLR4 protein expression in H9c2 cells, an effect mediated through the JNK pathway. Interestingly, E2 and/or ERα decreased the cytoplasmic HuR protein level and TLR4 mRNA stability, and farther decreased the level of TLR4 protein induced by LPS in H9c2 cardiomyoblast cells. Therefore, LPS triggered HuR expression which led to enhanced TLR4 mRNA and upregulated TLR4 expression through JNK1/2 in myocardial cells.
Assuntos
Proteínas ELAV/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/fisiologia , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/metabolismo , Estabilidade de RNA , Receptor 4 Toll-Like/genética , Animais , Células Cultivadas , Citoplasma/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Transporte Proteico/efeitos dos fármacos , Ratos , Fatores de TempoRESUMO
BACKGROUND: The c-Jun N-terminal kinase (JNK) is thought to be involved in inflammation, proliferation and apoptosis. AIM: To examine the role of JNK isoforms in metastasis, proliferation, migration and invasion of the malignant melanoma (MM) cell lines SK-MEL-28, SK-MEL-3 and WM164, using a kinase-specific inhibitor or isoform-specific small interfering (si)RNAs. RESULTS: SK-MEL-3, a cell line established from metastatic MM, showed slightly increased phosphorylation of both JNK1 and JNK2, whereas WM164, a cell line derived from primary MM, showed significant phosphorylation of JNK1. A JNK inhibitor, SP600125, inhibited cell proliferation of SK-MEL-3 but not SK-MEL-28 or WM164. Transfection of JNK1-specific siRNA reduced the migratory activity of WM164 cells, while silencing of either JNK1 or JNK2 strongly suppressed the invasive activity of SK-MEL-3. CONCLUSIONS: Our study suggests that JNK isoforms have different roles in MM. Metastasis of MM may be regulated by JNK2, while invasion is regulated by both JNK1 and JNK2. JNK1 and JNK2 respectively mediate cell migration and cell proliferation. Further understanding of the specific roles of JNK isoforms in the pathogenesis of MM may lead to the development of therapies targeting specific isoforms.
Assuntos
Melanoma/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Neoplasias Cutâneas/enzimologia , Antracenos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Melanoma/patologia , Invasividade Neoplásica , Isoformas de Proteínas/fisiologia , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Expression of WNT5A associated with aggressive tumor biology and poor clinical outcome of various types of cancer. However its function in the metastasis property of pancreatic cells still needs to be elucidated. METHODS: We detected the expressions of WNT5A, JNK1/p-JNK1 and Paxillin/p-Paxillin in cancer and the para-carcinoma tissues of pancreatic cancer. To understand how WNT5A/JNK signaling affects pancreatic cancer cell migration through the phosphorylation of cellular substrates of Paxillin, In vitro, we knocked down the WNT5A in PANC1, Capan-2 and HT1080 cell lines, and then tested the expression of JNK1. We detected the proteins of phosphorylation of Paxillin after JNK1 was inhibited and then the cells migration assay was evaluated. Moreover, JNK1 functionally phosphorylates serine178 on paxillin in vitro was detected .At last we subsequently observed whether WNT5A/JNK signaling modulates some molecule expressions relevant to focal adhesion (FA) formation and mesenchymal transition (EMT) and cell cycle. RESULTS: WNT5A, p-JNK1 and p-Paxillin were highly expressed in early stage of tumor tissues. In vitro, WNT5A/JNK signaling promotes cell migration in pancreatic cancer by phosphorylating serine178 on Paxillin, an FA adaptor, which means WNT5A may regulate FA's function.WNT5A up-regulates the molecule's expressions relevant to cell adhesion through the phosphorylation of JNK1, including MMP1, MMP2, ICAM and CD44. In addition, WNT5A/JNK signaling promoted the mRNA expressions of vimentin, but decreased in E-Cadherin expression, which suggested its regulatory effects on the EMT processes. WNT5A/JNK signaling didn't modulate cell proliferation. CONCLUSION: WNT5A/JNK signaling initiate cell migration of pancreatic cancer through activation of Paxillin, which suggested WNT5A has the potency of being an effective therapeutic target for the metastasis of pancreatic cancer.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Neoplasias Pancreáticas/fisiopatologia , Paxilina/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Adenocarcinoma/fisiopatologia , Linhagem Celular Tumoral , Humanos , Proteína Quinase 8 Ativada por Mitógeno/biossíntese , Paxilina/biossíntese , Fosforilação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Wnt/biossíntese , Proteína Wnt-5aRESUMO
We have identified the zinc-finger transcription factor Kruppel-like factor 4 (Klf4) among the transcription factors that are significantly downregulated in their expression during epithelial-mesenchymal transition (EMT) in mammary epithelial cells and in breast cancer cells. Loss and gain of function experiments demonstrate that the down-regulation of Klf4 expression is required for the induction of EMT in vitro and for metastasis in vivo. In addition, reduced Klf4 expression correlates with shorter disease-free survival of subsets of breast cancer patients. Yet, reduced expression of Klf4 also induces apoptosis in cells undergoing TGFß-induced EMT. Chromatin immunoprecipitation/deep-sequencing in combination with gene expression profiling reveals direct Klf4 target genes, including E-cadherin (Cdh1), N-cadherin (Cdh2), vimentin (Vim), ß-catenin (Ctnnb1), VEGF-A (Vegfa), endothelin-1 (Edn1) and Jnk1 (Mapk8). Thereby, Klf4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. Specifically, increased expression of Jnk1 (Mapk8) upon down-regulation of its transcriptional repressor Klf4 is required for EMT cell migration and for the induction of apoptosis. The data demonstrate a central role of Klf4 in the maintenance of epithelial cell differentiation and the prevention of EMT and metastasis.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Transativadores/fisiologia , Animais , Diferenciação Celular , Fator 4 Semelhante a Kruppel , Camundongos , Neoplasias Experimentais/patologiaRESUMO
c-Jun N-terminal kinases (JNKs) are central and ubiquitous mediators of cellular signaling for both physiogical-regenerative and pathological-apoptotic processes. Their impact on degeneration or inflammation is well documented, but so far little is known about their roles in higher brain functions. The more, the contribution of individual JNK isoforms remains obscure so far. Here we have tested the behaviour of JNK1, JNK2 and JNK3 knockout (ko) mice in elevated plus maze (EPM), open field (OF), novel object recognition memory (NORM) test and Morris water maze (MWM). Compared with wild type C57BL/6N mice JNK ko mice revealed significant differences. Taken together the data on anxiety, exploration and learning indicate that JNK1 ko mice displayed a stronger explorative behaviour and that knockout of JNK2 or JNK3 showed a tendency of behaviour opposite to that of JNK1 ko mice. This pattern reminds of the impact of individual JNK ko on neurodegeneration. This is the first comparative study on the impact of individual JNK ko on behavioural parameters.
Assuntos
Comportamento Animal/fisiologia , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Animais , Ansiedade/psicologia , Western Blotting , Peso Corporal/fisiologia , Encéfalo/enzimologia , Comportamento Exploratório/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Reconhecimento Psicológico/fisiologiaRESUMO
Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µM) or ERK1/2 (PD98059, 5 µM) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µM) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.