Palmitate induces SHIP2 expression via the ceramide-mediated activation of NF-κB,and JNK in skeletal muscle cells

Aims

Elevated plasma free fatty acids impair the insulin signaling by induction of the expression of protein phosphatases. However, the effect of palmitate on SH2-containing inositol 5′-phosphatase 2 (SHIP2) expression has not been investigated. Here we investigated the effects of palmitate on SHIP2 expression and elucidated the underlying mechanisms in skeletal muscle cells.

Main methods

SHIP2 mRNA and protein levels were measured in C2C12 myotubes exposed to palmitate. Specific inhibitors were used to identify the signaling pathways involved in SHIP2 expression.

Key findings

The results showed that 0.5 mM palmitate significantly upregulates the mRNA and protein levels of SHIP2 in C2C12 cells. To address the role of palmitate intracellular metabolites in SHIP2 expression, the myotubes were treated with palmitate in the presence of ceramide and diacylglycerol synthesis inhibitors. The results demonstrated that only ceramide synthesis inhibition could prevent palmitate-induced SHIP2 expression in these cells. In addition, the incubation of muscle cells with different concentrations of C2-ceramide dose-dependently enhanced SHIP2 expression. Furthermore, the inhibition of both JNK and NF-κB pathways could prevent ceramide-induced SHIP2 expression in myotubes.

Significance

These findings suggest that palmitate contributes to SHIP2 overexpression in skeletal muscle via the mechanisms involving the activation of ceramide-JNK and NF-κB pathways.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

Nna1 mediates Purkinje cell dendritic development via lysyl oxidase propeptide and NF-κB signaling

The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcd(Sid) mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcd(Sid) mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.     

BST2 promotes cell proliferation,migration and induces NF-κB activation in gastric cancer

Objectives

To investigate the functional roles of bone marrow stromal cell antigen 2 (BST2) in gastric cancer (GC) cells and its implications in the development of GC patients.

Results

BST2 was frequently overexpressed in GC tissues compared with the adjacent non-tumorous tissues, and high BST2 expression was correlated with tumor stage and lymphatic metastasis. Furthermore, in vitro experiments demonstrated that knockdown of BST2 by siRNA inhibited cell proliferation, induced apoptosis and repressed cell motility in GC cells. In addition, the pro-tumor function of BST2 in GC was mediated partly through the NF-κB signaling.

Conclusion

BST2 possesses the oncogenic potential in GC by regulating the proliferation, apoptosis, and migratory ability of GC cells, thereby BST2 could be a potential therapeutic target for the treatment of GC.

     

Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways

Objectives  

Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages.     

7-ketocholesterol induces apoptosis in differentiated PC12 cells via reactive oxygen species-dependent activation of NF-κB and Akt pathways

Cholesterol oxidation products formed under the enhanced oxidative stress in the brain are suggested to induce neuronal cell death. However, it is still unknown whether oxysterol-induced apoptosis in neuronal cells is mediated by Akt and NF-κB pathways. We assessed the apoptotic effect of 7-ketocholesterol against differentiated PC12 cells in relation to activation of the reactive oxygen species-dependent nuclear factor (NF)-κB, which is mediated by the Akt pathway. 7-Ketocholesterol induced a decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic Bax levels, cytochrome c release, caspase-3 activation and upregulation of p53. 7-Ketocholesterol induced an increase in phosphorylated inhibitory κB-α, NF-κB p65 and NF-κB p50 levels, binding of NF-κB p65 to DNA, and activation of Akt. Treatment with Bay 11-7085 (an inhibitor of NF-κB activation) and oxidant scavengers, including N-acetylcysteine, prevented the 7-ketocholesterol-induced formation of reactive oxygen species, activation of NF-κB, Akt and apoptosis-related proteins, and cell death. Results from this study suggest that 7-ketocholesterol may exert an apoptotic effect against PC12 cells by inducing activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell death pathway, leading to activation of caspases, via the reactive oxygen species-dependent activation of NF-κB, which is mediated by the Akt pathway.     

CARD4/Nod1 mediates NF-κB and JNK activation by invasive Shigella flexneri

Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-κB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-κB induction. Dominant-negative versions of CARD4 block activation of NF-κB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.     

Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.     

Apelin–APJ induces ICAM-1, VCAM-1 and MCP-1 expression via NF-κB/JNK signal pathway in human umbilical vein endothelial cells

Apelin receptor (APJ) deficiency has been reported to be preventive against atherosclerosis. However, the mechanism of this effect remains unknown. In this study, quantitative real-time RT-PCR, Western blotting and ELISA analyses revealed a significant increase in the expression of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) treated with apelin. Inhibitors of cellular signal transduction molecules were used to demonstrate involvement of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in apelin–APJ-induced activation of adhesion molecules and chemokines. Inhibition of APJ expression by RNA interference abrogated apelin-induced expression of adhesion molecules and chemokines and apelin-stimulated cellular signal transduction in HUVECs. The apelin–APJ system in endothelial cells is involved in the expression of adhesion molecules and chemokines, which are important for the initiation of endothelial inflammation-related atherosclerosis. Therefore, apelin–APJ and the cell signaling pathways activated by this system in endothelial cells may represent targets for therapy of atherosclerosis.     

Sanguinarine inhibits osteoclast formation and bone resorption via suppressing RANKL-induced activation of NF-κB and ERK signaling pathways

Sanguinarine is a natural plant extract that has been supplemented in a number of gingival health products to suppress the growth of dental plaque. However, whether sanguinarine has any effect on teeth and alveolar bone health is still unclear. In this study, we demonstrated for the first time that sanguinarine could suppress osteoclastic bone resorption and osteoclast formation in a dose-dependent manner. Sanguinarine diminished the expression of osteoclast marker genes, including TRAP, cathepsin K, calcitonin receptor, DC-STAMP, V-ATPase d2, NFATc1 and c-fos. Further investigation revealed that sanguinarine attenuated RANKL-mediated IκBα phosphorylation and degradation, leading to the impairment of NF-κB signaling pathway during osteoclast differentiation. In addition, sanguinarine also affected the ERK signaling pathway by inhibiting RANKL-induced ERK phosphorylation. Collectively, this study suggested that sanguinarine has protective effects on teeth and alveolar bone health.     

Leukotriene D4 induces amyloid-β generation via CysLT1R-mediated NF-κB pathways in primary neurons

Although the pathogenesis of sporadic Alzheimer’s disease (AD) is not clearly understood, neuroinflammation has been known to play a role in the pathogenesis of AD. To investigate a functional link between the neuroinflammation and AD, the effect of leukotriene D4 (LTD4), an inflammatory lipid mediator, was studied on amyloid-β generation in vitro. Application of LTD4 to cell monolayers at concentrations up to 40 nM LTD4 caused increases in the Aβ releases. Concentrations ?40 nM LTD4 decreased neuronal viability. Application of 20 nM LTD4 caused a significant increase in Aβ generation, as assessed by ELISA or Western blotting, without significant cytotoxicity. At this concentration, exposure of neurons to LTD4 for 24 h produced maximal effect in the Aβ generation, and significant increases in the expressions of cysteinyl leukotriene 1 receptor (CysLT₁R) and activity of β- or γ-secretase with complete abrogation by the selective CysLT₁R antagonist pranlukast. Exposure of neurons to LTD4 for 1 h showed activation of NF-κB pathway, by assessing the levels of p65 or phospho-p65 in the nucleus, and either CysLT₁R antagonist pranlukast or NF-κB inhibitor PDTC prevented the nuclear translocation of p65 and the consequent phosphorylation. PDTC also inhibited LTD4-induced elevations of β- or γ-secretase activity and Aβ generation in vitro. Overall, our data show for the first time that LTD4 causes Aβ production by enhancement of β- or γ-secretase resulting from activation of CysLT₁R-mediated NF-κB signaling pathway. These findings provide a novel pathologic link between neuroinflammation and AD.     

Chlamydia psittaci plasmid-encoded CPSIT_P7 induces macrophage polarization to enhance the antibacterial response through TLR4-mediated MAPK and NF-κB pathways

Although the protective effects of Chlamydia psittaci plasmid-encoded protein CPSIT_P7 as vaccine antigens to against chlamydial infection have been confirmed in our previous study, the function and mechanism of CPSIT_P7 inducing innate immunity in the antibacterial response remain unknown. Here, we found that plasmid protein CPSIT_P7 could induce M1 macrophage polarization upregulating the genes of the surface molecule CD86, proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and antibacterial effector NO synthase 2 (iNOS). During M1 macrophage polarization, macrophages acquire phagocytic and microbicidal competence, which promotes the host antibacterial response. As we observed that CPSIT_P7-induced M1 macrophages could partially reduce the infected mice pulmonary Chlamydia psittaci load. Furthermore, CPSIT_P7 induced M1 macrophage polarization through the TLR4-mediated MAPK and NF-κB pathways. Collectively, our results highlight the effect of CPSIT_P7 on macrophage polarization and provide new insights into new prevention and treatment strategies for chlamydial infection.     

Wnt5a activates THP-1 monocytic cells via a β-catenin-independent pathway involving JNK and NF-κB activation

Wnt5a has been implicated in the activation of macrophages. However, the profile and mechanism of downstream regulation has not been characterized. In this study, we have investigated the regulation of Wnt5a-induced activation in monocytic THP-1 cells. Wnt5a activated THP-1 cells, enhancing adhesion to endothelial cells. Hypoxia induced the production of Wnt5a, suggesting a role in the hypoxia-induced activation of macrophages. Wnt5a induced the expression of various pro-inflammatory cytokines and inflammatory mediators, particularly IL8 and CXCL2, suggesting a major role in the secretion of CXC chemokines by macrophages. Wnt5a induced JNK phosphorylation and NF-κB activation via β-catenin-independent signaling. Interestingly, SP600125, a specific inhibitor of JNK, inhibited Wnt5a-induced activation of NF-κB, supporting JNK-dependent NF-κB activation. Our data suggest that Wnt5a activates monocytic cells via JNK and NF-κB activation.     

Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS,PARP, IκBα/NF-κB,MAPKs, and mitochondria-dependent pathways: Prophylactic role of arjunolic acid

Diabetic mellitus, a chronic metabolic disorder, is one of the most important health problems in the world, especially in developing countries. Our earlier investigations reported the beneficial action of arjunolic acid (AA) against streptozotocin-mediated type 1 hyperglycemia. We have demonstrated that AA possesses protective roles against drug- and chemical- (environmental toxins) induced hepatotoxicity. Liver is the main organ of detoxification. The purpose of this study was to explore whether AA plays any protective role against hyperglycemic hepatic dysfunctions and, if so, what molecular pathways it utilizes for the mechanism of its protective action. In experimental rats, type 1 hyperglycemia was induced by streptozotocin. AA was administered orally at a dose of 20 mg/kg body wt both before and after diabetic induction. An insulin-treated group was included in the study as a positive control for type 1 diabetes. Hyperglycemia caused a loss in body weight, reduction in serum insulin level, and increased formation of HbA_1C as well as advanced glycation end products (AGEs). Elevated levels of serum ALT and ALP, increased production of ROS and RNS, increased lipid peroxidation, increased 8-OHdG/2-dG ratio, and decreased GSH content and cellular antioxidant defense established the hyperglycemic liver dysfunction. Activation of iNOS, IκBα/NF-κB, and MAPK pathways as well as signals from mitochondria were found to be involved in initiating apoptotic cell death. Hyperglycemia caused overexpression of PARP, reduction in intracellular NAD as well as ATP level, and increased DNA fragmentation in the liver tissue of the diabetic animals. Results of immunofluorescence (using anti-caspase-3 and anti-Apaf-1 antibodies), DAPI/PI staining, and DNA ladder formation and information obtained from FACS analysis confirmed the apoptotic cell death in diabetic liver tissue. Histological studies also support the experimental findings. AA treatment prevented or ameliorated the diabetic liver complications and apoptotic cell death. The effectiveness of AA in preventing the formation of ROS, RNS, HbA_1C, AGEs, and oxidative stress signaling cascades and protecting against PARP-mediated DNA fragmentation can speak about its potential uses for diabetic patients.