Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways

Over‐activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti‐inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose‐dependent manner, significantly decreased osteoclast‐related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor‐kappaB (NF‐κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency‐induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down‐regulating the NF‐κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Cell entry of bovine ephemeral fever virus requires activation of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways as well as Cox‐2‐mediated PGE2/EP receptor signalling to enhance clathrin‐mediated virus endocytosis

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin‐mediated and dynamin 2‐dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src‐JNK signalling to enhance its entry. Activation of these pathways by ultraviolet‐inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV‐induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3′‐dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox‐2‐catalysed prostaglandin E₂ (PGE₂) synthesis and induces expressions of G‐protein‐coupled E‐prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV‐induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox‐2‐mediated PGE₂/EP receptor signalling pathways, further enhancing Src‐JNK‐AP1 in a cAMP‐dependent manner and PI3K‐Akt‐NF‐κB in a cAMP‐independent manner. Accordingly, BEFV stimulates PGE₂/EP receptor signalling amplifying Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways in an autocrine or paracrine fashion to enhance virus entry.     

CUDC‐907 blocks multiple pro‐survival signals and abrogates microenvironment protection in CLL

CUDC‐907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in hematopoietic malignancies. However, the molecular mechanisms of its effects in chronic lymphocytic leukaemia (CLL) remain elusive. We show that CLL cells are sensitive to CUDC‐907, even under conditions similar to the protective microenvironment of proliferation centres. CUDC‐907 inhibited PI3K/AKT and HDAC activity, as expected, but also suppressed RAF/MEK/ERK and STAT3 signalling and reduced the expression of anti‐apoptotic BCL‐2 family proteins BCL‐2, BCL‐xL, and MCL‐1. Moreover, CUDC‐907 downregulated cytokines BAFF and APRIL and their receptors BAFFR, TACI, and BCMA, thus blocking BAFF‐induced NF‐κB signalling. T cell chemokines CCL3/4/17/22 and phosphorylation of CXCR4 were also reduced by CUDC‐907. These data indicated that CUDC‐907 abrogates different protective signals and suggested that it might sensitize CLL cells to other drugs. Indeed, combinations of low concentrations of CUDC‐907 with inhibitors of BCL2, BTK, or the NF‐κB pathway showed a potent synergistic effect. Our data indicate that, apart from its known functions, CUDC‐907 blocks multiple pro‐survival pathways to overcome microenvironment protection in CLL cells. This provides a rationale to evaluate the clinical relevance of CUDC‐907 in combination therapies with other targeted inhibitors.     

p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

Cullin‐RING‐ubiquitin‐ligase (CRL)‐dependent ubiquitination of the nuclear factor kappa B (NF‐κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF‐κB/RelA nuclear activity. Through removal of the CRL‐activating modification of their cullin subunit with the ubiquitin (Ub)‐like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub‐ligase activity. While RelA phosphorylation was observed to mediate NF‐κB activation independent of Ub‐proteasome‐pathway (UPP)‐dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF‐κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)‐induced NF‐κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK‐complex‐dependent manner within the NF‐κB/RelA‐IκBα‐complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF‐κB/RelA from IκBα. Subsequent to CRL‐dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP‐promoted timely and efficient degradation of IκBα as well as simultaneous NF‐κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP‐supported scheduled basal NF‐κB activity, and the mechanism of TNF‐induced NF‐κB activation.     

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K,PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L ‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O, H₂O₂, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009. © 2009 Wiley‐Liss, Inc.     

FAK activation is required for TNF‐α‐induced IL‐6 production in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. IL‐6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL‐6 production stimulated by TNF‐α in cultured myoblasts. TNF‐α caused concentration‐dependent increases in IL‐6 production. TNF‐α‐mediated IL‐6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3‐kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF‐κB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IκB protease inhibitor (L ‐1‐tosylamido‐2‐phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF‐α. TNF‐α increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF‐α activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, p65 phosphorylation, and κB‐luciferase activity. TNF‐α mediated an increase of κB‐luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF‐α increased IL‐6 production in myoblasts via the FAK/PI3K/Akt and NF‐κB signaling pathway. J. Cell. Physiol. 223: 389–396, 2010. © 2010 Wiley‐Liss, Inc.     

Hypothalamic POMC expression is required for peripheral insulin action on hepatic gluconeogenesis through regulating STAT3 in sepsis rats

Liver injury and dysregulated glucose homoeostasis are common manifestations during sepsis. Although plenty of studies reported insulin could protect against multiple organ injuries caused by critical infections among patients, little was known about the precise mechanism. We investigated whether liver inflammatory pathway and central neuropeptides were involved in the process. In sepsis rats, hepatic IKK/NF‐κB pathway and STAT3 were strongly activated, along with reduced body weight, blood glucose and suppressed hepatic gluconeogenesis (GNG). Peripheral insulin administration efficiently attenuated liver dysfunction and glucose metabolic disorders by suppressing hypothalamic anorexigenic neuropeptide proopiomelanocortin (POMC) expression, hepatic NF‐κB pathway and STAT3 phosphorylation. Furthermore, knockdown of hypothalamic POMC significantly diminished protective effect of insulin on hepatic GNG and insulin‐induced STAT3 inactivation, but not inflammation or IKK/NF‐κB pathway. These results suggest that hepatic IKK/NF‐κB pathway mediates the anti‐inflammatory effect of insulin in septic rats, and peripheral insulin treatment may improve hepatic GNG by inhibiting STAT3 phosphorylation dependent on hypothalamic POMC expression.     

Interleukin-11 increases cell motility and up-regulates intercellular adhesion molecule-1 expression in human chondrosarcoma cells

Interleukin‐11 (IL‐11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL‐11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL‐11 on human chondrosarcoma cells are largely unknown. Here, we found that IL‐11 increased the migration and expression of intercellular adhesion molecule‐1 (ICAM)‐1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL‐11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB pathways were activated by IL‐11 treatment, and the IL‐11‐induced expression of ICAM‐1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF‐κB cascades. Taken together, our results indicate that IL‐11 enhanced the migration of the chondrosarcoma cells by increasing ICAM‐1 expression through the IL‐11Rα receptor, PI3K, Akt, and NF‐κB signal transduction pathway. J. Cell. Biochem. 113: 3353–3362, 2012. © 2012 Wiley Periodicals, Inc.     

Hyperglycemia induces NF‐κB activation and MCP‐1 expression via downregulating GLP‐1R expression in rat mesangial cells: inhibition by metformin

Hyperglycemia impairs glucagon‐like peptide‐1 receptor (GLP‐1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP‐1R agonists. We hypothesized that the downregulation of GLP‐1R by hyperglycemia might reduce the renal‐protective effects of GLP‐1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP‐1R and its signaling pathways in the HBZY‐1 rat mesangial cell line. We found that high glucose reduced GLP‐1R messenger RNA (mRNA) levels in HBZY‐1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP‐1R agonist exendin‐4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti‐inflammatory functions in HBZY‐1 cells linked with nuclear factor‐κB (NF‐κB) activation. In consistence, GLP‐1R inhibition aggravated the high glucose‐induced activation of NF‐κB and MCP‐1 protein levels in cultured HBZY‐1 cells while overexpression of GLP‐1R opposite effects. We further proved that metformin restored high glucose‐inhibited GLP‐1R mRNA expression and decreased high glucose evoked inflammation in HBZY‐1 cells. On the basis of these findings, we conclude that high glucose lowers GLP‐1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP‐1R agonists in DN.     

NF‐κB/NKILA signaling modulates the anti‐cancerous effects of EZH2 inhibition

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.