RESUMEN
OBJECTIVES: Sanshimao (SSM) is a traditional Chinese medicine formula for advanced hepatocellular carcinoma (HCC). This study was designed to investigate the effect of SSM on HCC-induced angiogenesis and to explore the potential mechanism. METHODS: The endothelial cells were cultured with HCC cells conditioned medium in the 1% oxygen atmosphere to imitate tumor hypoxia microenvironment. EA.hy926 cells migration and tubulogenesis were detected by tube formation and scratch-wound assay. The protein microarray was employed to explore SSM-targeted proteins in Huh7 cells. We also established an animal model to observe the effects of SSM on angiogenesis in vivo. RESULTS: The data indicated that SSM reduced HCC-induced migration and tube formation of EA.hy926 cells at low dose under hypoxic conditions. These effects might be partly owing to suppression of HIF-1α-induced vascular endothelial growth factorα expression in Huh7 cells. Moreover, this inhibition was in an MKK6/P38-dependent way. Besides, Huh7 subcutaneous tumor models in nude mice further demonstrated the inhibition of SSM on tumor weight might be exerted partly by reduction of angiogenesis via blocking MKK6/P38 signaling pathways. CONCLUSION: SSM inhibits HCC-induced pro-angiogenesis under hypoxic conditions via suppression of MKK6/P38 signaling pathways, which is favorable for HCC tumor growth.
Asunto(s)
Carcinoma Hepatocelular , Medicamentos Herbarios Chinos , Neoplasias Hepáticas , Neovascularización Patológica , Animales , Ratones , Angiogénesis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Células Endoteliales/metabolismo , Hipoxia/tratamiento farmacológico , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Transducción de Señal , Microambiente Tumoral , Medicamentos Herbarios Chinos/farmacología , MAP Quinasa Quinasa 6/efectos de los fármacos , MAP Quinasa Quinasa 6/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Apurinic/apyrimidinic endonuclease 1 (APEX1), a key enzyme responsible for DNA base excision repair, has been linked to development and progression of cancers. In this work, we aimed to explore the role of APEX1 in hepatocellular carcinoma (HCC) and elucidate its molecular mechanism. METHODS: The expression of APEX1 in HCC tissues and matched adjacent normal tissues (n = 80 cases) was evaluated by immunohistochemistry. Web-based tools UALCAN and the Kaplan-Meier plotter were used to analyze the Cancer Genome Atlas database to compare expression of APEX1 mRNA to 5-year overall survival. APEX1 was stably silenced in two HCC cell lines, Hep 3B and Bel-7402, with shRNA technology. An in vivo tumorigenesis model was established by subcutaneously injecting sh-APEX1-transfected Bel-7402 cells into mice, and tumor growth was determined. We performed high-throughput transcriptome sequencing in sh-APEX1-treated HCC cells to identify the key KEGG signaling pathways induced by silencing of APEX1. RESULTS: APEX1 was significantly upregulated and predicted poor clinical overall survival in HCC patients. Silencing APEX1 inhibited the proliferation of HCC cells in vivo and in vitro, and it repressed invasion and migration and increased apoptosis and the percentage of cells in G1. Differentially expressed genes upon APEX1 silencing included genes involved in TNF signaling. A positive correlation between the expression of APEX1 and MAP2K6 was noted, and overexpressing MAP2K6 overcame cancer-related phenotypes associated with APEX1 silencing. CONCLUSION: APEX1 enhances the malignant properties of HCC via MAP2K6. APEX1 may represent a valuable prognostic biomarker and therapeutic target in HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratones , Biomarcadores , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/genética , ADN , Endonucleasas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , ARN Mensajero , ARN Interferente Pequeño/genética , HumanosRESUMEN
Fenbendazole, a broad-spectrum anti-parasitic drug, can be a potential anti-tumor agent. In this study, we synthesized and purified its derivative, analog 6, intending to achieve improved efficacy in cancer cells and decreased toxicity in normal cells. To evaluate in vitro anti-tumor activities of fenbendazole and analog 6 in different cancer cell lines, a CCK-8 assay was performed, and we found that human cervical cancer HeLa cells were more sensitive to analog 6 than to fenbendazole. Furthermore, we explored the associated mechanism, and our results showed that analog 6 and fenbendazole could induce oxidative stress by accumulating ROS. It not only activated the p38-MAPK signaling pathway, thereby inhibiting the proliferation of HeLa cells and enhancing the apoptosis of HeLa cells, but also significantly induced impaired energy metabolism and restrained their migration and invasion. In addition, the modified analog 6 showed reduced toxicity to normal cells without decreased anti-cancer effect. In conclusion, fenbendazole and analog 6 have multiple targets and strong anti-tumor effects on HeLa cells in vitro and in vivo. The optimized analog 6 could inhibit the viability of HeLa cells with lower toxicity than normal human cells, promising to be developed as an antitumor active compound.
Asunto(s)
Neoplasias del Cuello Uterino , Proteínas Quinasas p38 Activadas por Mitógenos , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Metabolismo Energético , Femenino , Fenbendazol/farmacología , Células HeLa , Humanos , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Estrés Oxidativo , Neoplasias del Cuello Uterino/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Helicobacter pylori infection is a leading cause of gastric cancer (GC). However, the underlying mechanisms have not yet been fully elucidated. We aimed to identify microRNAs (miRNAs) regulated by H. pylori infection and their underlying mechanisms in gastric carcinogenesis. Using a mouse model, it was established that H. pylori infection inhibited autophagy in the gastric mucosa. Importantly, H. pylori infection decreased miR-1298-5p levels in human and mouse gastric tissues and human gastric cell lines. Furthermore, the downregulation of miR-1298-5p levels remarkably inhibited autophagy, ultimately increasing the intracellular H. pylori load, which was detected using a gentamicin protection assay. A series of in vitro assays showed that the downregulation of miR-1298-5p expression promoted GC cell proliferation, migration, and invasion. Mechanistically, using bioinformatics prediction, miRNA pull-down assays, and luciferase reporter assays, mitogen-activated protein kinase kinase 6 (MAP2K6) was found to be the direct target of miR-1298-5p, through which miR-1298-5p regulated autophagy and GC cell viability and motility. Moreover, MAP2K6/p38 mitogen-activated protein kinase (MAPK) axis was determined to be the downstream pathway of miR-1298-5p. These findings revealed that H. pylori infection was found to inhibit autophagy and promote tumor growth by regulating miR-1298-5p expression and the miR-1298-5p/MAP2K6/p38 MAPK axis might be a new avenue for the clinical management of H. pylori infection and H. pylori-associated GC.
Asunto(s)
Autofagia , Infecciones por Helicobacter , MAP Quinasa Quinasa 6 , MicroARNs , Neoplasias Gástricas , Autofagia/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismo , Humanos , MAP Quinasa Quinasa 6/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes.
Asunto(s)
Cromatina , Heterocromatina , Reprogramación Celular , Epigénesis Genética , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , FosforilaciónRESUMEN
BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is highly associated with the progression and metastasis of various solid tumours. MAPK14, a core molecule of the MAPK pathway, plays vital roles in the colorectal cancer (CRC). Recent studies have shown that circRNAs can affect tumour progression by encoding peptides. However, little is known regarding the potential protein translated from circMAPK14 and whether it plays a role in the carcinogenesis of colorectal cancer. METHODS: The RNA level and translatable potential of circMAPK14 in CRC was verified using qRT-PCR and public databases. RNase R digestion assay, qRT-PCR, sanger sequencing and FISH assays were utilised to verify the circular characteristics and subcellular localisation of circMAPK14. The suppressive role of circMAPK14 on the progression and metastasis of CRC was verified in vivo and in vitro. LC/MS analysis combined with western blotting demonstrated the presence and relative expression of circMAPK14-175aa. The underlying mechanism of circMAPK14-175aa action to inhibit CRC was identified by co-IP analysis. The binding of U2AF2 within the flanking introns of circMAPK14 was evaluated by RNA pull-down assay and RIP assay. Ultimately, luciferase reporter gene assays and ChIP assays confirmed that FOXC1 suppressed transcription of U2AF2 by binding to the U2AF2 promoter in the -400 bp to -100 bp region.ã RESULTS: We identified that hsa_circ_0131663 (termed circMAPK14) showed significantly decreased expression level in cells and tissue samples of CRC, and was primarily localised in the cytoplasm. A series of function experiments demonstrated that circMAPK14 influenced CRC progression and metastasis by encoding a peptide of 175 amino acids (termed circMAPK14-175aa). We also found that circMAPK14-175aa reduced nuclear translocation of MAPK14 by competitively binding to MKK6, thus facilitating ubiquitin-mediated degradation of FOXC1. Moreover, we described a positive feedback loop in CRC in which elevated FOXC1 expression was caused by reduced circMAPK14-175aa expression. This, in turn, decreased circMAPK14 biogenesis by suppressing U2AF2 transcription. CONCLUSION: In summary, we reported for the first time that circMAPK14 functioned as a tumour-suppressor by encoding circMAPK14-175aa, which blocked the progression and metastasis of colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Animales , Biomarcadores de Tumor/genética , Proliferación Celular/genética , Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/metabolismo , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Genes Supresores de Tumor , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disease characterized by deterioration of articular cartilage functions. Previous studies have confirmed the role of circular RNAs (circRNAs) in OA, but the role of mechanical stress-related circRNA (circRNA-MSR) in OA is unknown. DESIGN: The human chondrocytes C28/I2 were cultured and treated with lipopolysaccharide (LPS) to establish the OA model. The mRNA and protein levels were measured by qRT-PCR or Western blot. Cell viability was analyzed by MTT assay. Flow cytometry was carried out to detect cell apoptosis. The levels of TNF-α, IL-1ß, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Pull-down assay was conducted to measure circRNA-MSR-related miRNA. Dual-luciferase reporter gene detection was performed to detect the target relationships between miR-643 and circRNA-MSR or Mitogen-activated protein kinase kinase 6 (MAP2K6). The RNA-fluorescence in situ hybridization (RNA-FISH) assay was conducted to verify the localization of circRNA-MSR and miR-643. RESULTS: The expressions of circRNA-MSR were upregulated in LPS stimulated C28/I2 cells. Knockdown of circRNA-MSR can inhibit LPS-induced apoptosis, inflammatory response, and extracellular matrix (ECM) degradation, and promote cell C28/I2 cells proliferation. Moreover, circRNA-MSR directly targeted miR-643. RNA-FISH exhibited that circRNA-MSR may act as a competing endogenous RNA (ceRNA) of miR-643. Over-expression of miR-643 could alleviate LPS-induced C28/I2 chondrocyte injury and promote cell proliferation. Besides, miR-643 directly bound to MAP2K6 mRNA. MiR-643 inhibition or MAP2K6 overexpression can reverse the role of circRNA-MSR knockdown on LPS-treated chondrocytes. CONCLUSION: circRNA-MSR can upregulate MAP2K6 by targeting miR-643, thereby inhibiting cell proliferation and promoting apoptosis of C28/I2 cells.
Asunto(s)
Cartílago Articular , MicroARNs , Osteoartritis , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , MicroARNs/genética , Osteoartritis/genética , Osteoartritis/metabolismo , ARN Circular/genética , Transducción de SeñalRESUMEN
Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn-/- cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn-/- cells and could serve as basis for further research.
Asunto(s)
Apoptosis , Citoprotección , Miostatina/deficiencia , Estrés Oxidativo , Aldehídos/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Movimiento Celular , Proliferación Celular , Replicación del ADN , Peroxidación de Lípido , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Ratones , Miostatina/metabolismo , Estrés Nitrosativo , Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
To investigate whether the beiging process changes the interactive effects of salt and MEK6 gene on inflammatory adipogenesis, the salt treatment (NaCl 50 mM) and MEK6 transfection of Tg(+/+) cells were performed with white adipocytes (WAT) and beige-like-adipocytes (BLA). BLA induced by T3 were confirmed by UCP-1 expression and the MEK6 protein was 3.5 times higher in MEK6 transfected WAT than the control. The adipogenic genes, PPAR-γ and C/EBP-α, were 1.5 times more highly expressed in the salt-treated groups than the non-salt-treated groups, and adipogenesis was greatly increased in Tg(+/+) WAT compared to non-transfected Tg(-/-). The adipogenesis induced by salt treatment and MEK6 transfection was significantly reduced in BLA. The inflammatory adipocytokines, TNF-α, IL-1ß, and IL-6, were increased in the salt-treated Tg(+/+) WAT, but an anti-inflammation biomarker, the adiponectin/leptin ratio, was reduced in Tg(+/+), to tenth of that in Tg(-/-). However, the production of adipocytokines in WAT was strongly weakened in BLA, although a combination of salt and MEK6 transfection had the most significant effects on inflammation in both WAT and BLA. Oxygen consumption in mitochondria was maximized in salt-treated and MEK6 transfected WAT, but it was decreased by 50% in BLA. In conclusion, beiging controls the synergistic effects of salt and MEK6 on adipogenesis, inflammation, and energy expenditure.
Asunto(s)
Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Adipogénesis , MAP Quinasa Quinasa 6/metabolismo , Cloruro de Sodio/farmacología , Células 3T3-L1 , Adipocitos Beige/citología , Adipocitos Blancos/citología , Adipocitos Blancos/efectos de los fármacos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Metabolismo Energético , MAP Quinasa Quinasa 6/genética , Ratones , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.
Asunto(s)
MAP Quinasa Quinasa 6/química , Proteínas Quinasas p38 Activadas por Mitógenos/química , Sitios de Unión , Activación Enzimática , Humanos , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Acute pancreatitis (AP) is a common acute abdominal disease with high mortality and mortality rates. Increasing evidences clarified that Traditional Chinese Medicine (TCM) adjuvant therapy for AP can be used and it gives a positive effect. Quercetin (3,3',4',5,7-pentahydroxyflavone, QE) is a type of flavone compound with positive effect on cancer and inflammation prevention. The current study aims to identify the effect of QE on AP and potential molecular effect. In this case, caerulein (CAE) induced AP cell and mice model were used. QE alleviated inflammatory mediators TNF-α, IL-6, and IL-10 in experiments. In addition, miR-216b was increased based on QE treatment. In further study, MAP2K6 of p38/MAPK signaling pathway was identified as a direct target of miR-216b, and QE inhibited p38/MAPK signaling pathway through up-regulating miR-216b. Our study also first confirmed that long non-coding RNA NEAT1 is a direct target of miR-216b and can be suppressed by QE. Because of the target, NEAT1, miR-216b, and MAP2K6 formed a competitive endogenous RNA (ceRNA) network. Besides direct target mediated by QE, it also decreased TNF-α which down-regulated TRAF2 and MAP3K5 located on upstream of p38/MAPK signaling and formed a feedback loop. In conclusion, QE has a protective effect on AP through inhibiting p38/MAPK signaling pathway by up-regulating miR-216b and suppressing TNF-α.
Asunto(s)
MicroARNs/genética , Páncreas/efectos de los fármacos , Pancreatitis/prevención & control , Quercetina/farmacología , Animales , Línea Celular , Ceruletida/toxicidad , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , MAP Quinasa Quinasa 6/metabolismo , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Páncreas/patología , ARN Largo no Codificante/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
miRNAs are a small class of noncoding RNAs that perform biological functions by regulating the stability or translation of target genes in various biological processes. This study illustrated the role of miR-10a-5p, which is relatively enriched in adipose tissues, using primary mouse preadipocytes as model. With elevated miR-10a-5p expression, the proliferative ability of mouse preadipocytes was significantly enhanced, indicated by increased EdU+ cells and G1/S transition, accompanied by upregulated Cyclin B, Cyclin D and PCNA and downregulated p21 and p27. Meanwhile, the adipogenic differentiation was significantly attenuated by elevated miR-10a-5p, supported by Oil Red O staining and suppressed PPARγ and aP2 expression. Furthermore, Map2k6 and Fasn were predicted to be the target genes of miR-10a-5p in silico, and dual luciferase reporter assay confirmed the direct targeting effects. Western blot analysis results showed that miR-10a-5p specially reduced Map2k6 expression at the proliferative stage without affecting Fasn expression, while significantly restrained Fasn expression with unchanged Map2k6 expression during adipogenic differentiation. Taken together, these results revealed a potential role of miR-10a-5p in adipogenesis and in the treatment of obesity.
Asunto(s)
Adipogénesis/genética , Acido Graso Sintasa Tipo I/metabolismo , MAP Quinasa Quinasa 6/metabolismo , MicroARNs/biosíntesis , MicroARNs/genética , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Ratones Endogámicos C57BL , Cultivo Primario de Células , Regulación hacia ArribaRESUMEN
The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.
Asunto(s)
MAP Quinasa Quinasa 6/metabolismo , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Animales , Apoptosis/efectos de la radiación , Línea Celular , Proliferación Celular/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , MAP Quinasa Quinasa 6/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutagénesis , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.
Asunto(s)
Quimiocina CCL20/metabolismo , Células Estrelladas Hepáticas/metabolismo , Nicotinamida Fosforribosiltransferasa/farmacología , Quimiocina CCL20/fisiología , Quimiocinas/metabolismo , Hepatocitos/metabolismo , Humanos , Janus Quinasa 2/metabolismo , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Macrófagos/metabolismo , FN-kappa B/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Células THP-1 , Factor de Transcripción ReIA/metabolismoRESUMEN
Nutrients are absorbed solely by the intestinal villi. Aging of this organ causes malabsorption and associated illnesses, yet its aging mechanisms remain unclear. Here, we show that aging-caused intestinal villus structural and functional decline is regulated by mTORC1, a sensor of nutrients and growth factors, which is highly activated in intestinal stem and progenitor cells in geriatric mice. These aging phenotypes are recapitulated in intestinal stem cell-specific Tsc1 knockout mice. Mechanistically, mTORC1 activation increases protein synthesis of MKK6 and augments activation of the p38 MAPK-p53 pathway, leading to decreases in the number and activity of intestinal stem cells as well as villus size and density. Targeting p38 MAPK or p53 prevents or rescues ISC and villus aging and nutrient absorption defects. These findings reveal that mTORC1 drives aging by augmenting a prominent stress response pathway in gut stem cells and identify p38 MAPK as an anti-aging target downstream of mTORC1.
Asunto(s)
Intestinos/citología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células Madre/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Envejecimiento , Animales , Proliferación Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/fisiología , MAP Quinasa Quinasa 6/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones Noqueados , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Sirolimus/farmacología , Células Madre/citología , Tamoxifeno/farmacología , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genes jun/genética , Humanos , Interleucina-1beta/farmacología , Interleucina-8/genética , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/genética , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Mutación Puntual , Regiones Promotoras Genéticas , Eliminación de Secuencia , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Stroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown. METHODS: Mice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22â¯cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B-I, LC3B-II, Beclin 1, and p62 expression. RESULTS: Significantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22â¯cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B-I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719. CONCLUSIONS: Our results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.
Asunto(s)
Apoptosis , MAP Quinasa Quinasa 6/metabolismo , MicroARNs/metabolismo , Neuronas/metabolismo , Neuronas/patología , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Animales , Autofagia , Secuencia de Bases , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glucosa , MAP Quinasa Quinasa 6/genética , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Neuronas/ultraestructura , Oxígeno , ARN Circular , Accidente Cerebrovascular/genéticaRESUMEN
BACKGROUND: Oligomeric amyloid-ß peptide (Aß) is associated with dysfunctional neuronal networks and neuronal loss in the development of Alzheimer's disease (AD). Ischemic postconditioning protects against post-ischemic excitotoxicity, oxidative stress, and inflammatory process that have also been implicated in the pathogenesis of AD. Evaluating the roles of ischemic postconditioning in oligomeric Aß-induced neurotoxicity and underlying signal events may provide potential strategy for medical therapy in AD. OBJECTIVES: The aim of the present study was to explore whether and how a brief ischemic postconditioning protects against Aß neurotoxicity in rat hippocampus. METHODS: Oligomeric Aß25-35 (20 nmol/rat) or Aß1-42 (5 nmol/rat) was infused by intracerebroventricular injection in adult male Sprague-Dawley rats. Ischemic postconditioning, a brief episode of global brain ischemia (3âmin), was conducted at 1, 3, or 7 days after Aß treatment, respectively. RESULTS: A brief ischemic postconditioning reduced neuronal loss and inhibited the activation of MLK3, MKK3/6, and P38MAPKs in rat hippocampal CA1 and CA3 subfields after Aß oligomer infusion. An N-methyl-D-aspartate (NMDA) receptor antagonist amantadine, but not non-NMDA receptor antagonist CNQX, reversed the MLK3-MKK3/6-P38MAPK signal events and beneficial effect of ischemic postconditioning on neuronal survival. Such reversion was also realized by NVP-AAM077, a GluN2A-subunit-selective NMDA receptor antagonist. Moreover, posttreatment with low doses of NMDA (5 nmol-40 nmol/rat) suppressed the Aß-induced P38MAPK signaling and imitated the neuroprotection of ischemic postconditioning against Aß neurotoxicity. CONCLUSIONS: Ischemic postconditioning provides neuroprotection against Aß neurotoxicity by moderate upregulation of NMDA receptor signaling, especially GluN2A-containing NMDA receptor pathway, and thereafter downregulation of MLK3-MKK3/6-P38MAPK signal events.
Asunto(s)
Hipocampo/metabolismo , Poscondicionamiento Isquémico/métodos , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Hipocampo/efectos de los fármacos , MAP Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 6/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Masculino , Fragmentos de Péptidos/toxicidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
Reprogramming somatic cells to pluripotency by Oct4, Sox2, Klf4, and Myc represent a paradigm for cell fate determination. Here, we report a combination of Jdp2, Jhdm1b, Mkk6, Glis1, Nanog, Essrb, and Sall4 (7F) that reprogram mouse embryonic fibroblasts or MEFs to chimera competent induced pluripotent stem cells (iPSCs) efficiently. RNA sequencing (RNA-seq) and ATAC-seq reveal distinct mechanisms for 7F induction of pluripotency. Dropout experiments further reveal a highly cooperative process among 7F to dynamically close and open chromatin loci that encode a network of transcription factors to mediate reprogramming. These results establish an alternative paradigm for reprogramming that may be useful for analyzing cell fate control.
Asunto(s)
Reprogramación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/metabolismo , Células Madre Pluripotentes Inducidas/citología , Histona Demetilasas con Dominio de Jumonji/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Proteína Homeótica Nanog/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Quimera/genética , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Eucromatina/genética , Eucromatina/metabolismo , Proteínas F-Box/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Factor 4 Similar a Kruppel , MAP Quinasa Quinasa 6/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Homeótica Nanog/genética , RNA-Seq , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND The objective of this study was to study the anti-inflammatory effect and possibly involved molecular mechanisms of matrine on oxidized low-density lipoprotein (ox-LDL)-exposed macrophages. MATERIAL AND METHODS Cultured human macrophages (THP-1 cell line) were exposed to ox-LDL at final concentrations of 0, 25, 50, and 100 µg/mL. Several cells were then treated with matrine at serial diluted concentrations. 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining was used to evaluate reactive oxygen species (ROS) production; a colorimetric method was used to determine the cellular antioxidant capacity; production of pro-inflammatory cytokines interleukin (IL)18 and tumor necrosis factor (TNF)alpha were determined by enzyme-linked immunosorbent assay (ELISA); and immunoblot assay was used to assess the relative protein phosphorylation and expression. RESULTS ox-LDL exposure significantly elevated intracellular ROS level and supernatant IL18 and TNFalpha concentrations, but impaired total antioxidant capacity (TAC) of macrophages. The relative phosphorylations of MAPK kinase kinases (MKK)6, MKK3, and p38 mitogen-activated protein kinases (MAPK) were increased by ox-LDL exposure. The expression levels of IL18 and TNFalpha were also increased in ox-LDL-treated macrophages. The matrine treatment reduced intracellular ROS level and supernatant IL18 and TNFalpha concentrations and increased TAC in a concentration- dependent manner. The relative phosphorylations of MKK6, MKK3, and p38 MAPK were reduced after matrine administration. Moreover, the expression levels of IL18 and TNFalpha were also decreased by matrine treatment, in a concentration-dependent manner. CONCLUSIONS ox-LDL increases inflammatory response in macrophages by activating the ROS-mediated MKKs/p38 MAPK-induced inflammatory signaling pathway. Matrine suppresses ox-LDL-induced inflammatory by inhibiting the MKKs/p38 MAPK signaling pathway.