Activation of the JNK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on CXCR4 and CD74

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.     

Cyclic Mechanical Stretch Decreases Cell Migration by Inhibiting Phosphatidylinositol 3-Kinase- and Focal Adhesion Kinase-mediated JNK1 Activation

Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.     

Chemical Genomic-Based Pathway Analyses for Epidermal Growth Factor-Mediated Signaling in Migrating Cancer Cells

To explore the diversity and consistency of the signaling pathways that regulate tumor cell migration, we chose three human tumor cell lines that migrated after treatment with EGF. We then quantified the effect of fifteen inhibitors on the levels of expression or the phosphorylation levels of nine proteins that were induced by EGF stimulation in each of these cell lines. Based on the data obtained in this study and chemical-biological assumptions, we deduced cell migration pathways in each tumor cell line, and then compared them. As a result, we found that both the MEK/ERK and JNK/c-Jun pathways were activated in all three migrating cell lines. Moreover, GSK-3 and p38 were found to regulate PI3K/Akt pathway in only EC109 cells, and JNK was found to crosstalk with p38 and Fos related pathway in only TT cells. Taken together, our analytical system could easily distinguish between the common and cell type-specific pathways responsible for tumor cell migration.     

Daunorubicin induces cell death via activation of apoptotic signalling pathway and inactivation of survival pathway in muscle-derived stem cells

Daunorubicin (as well as other anthracyclines) is known to be toxic to heart cells and other cells in organism thus limiting its applicability in human cancer therapy. To investigate possible mechanisms of daunorubicin cytotoxicity, we used stem cell lines derived from adult rabbit skeletal muscle. Recently, we have shown that daunorubicin induces apoptotic cell death in our cell model system and distinctly influences the activity of MAP kinases. Here, we demonstrate that two widely accepted antagonistic signalling pathways namely proapoptotic JNK and prosurvival PI3K/AKT participate in apoptosis. Using the Western blot method, we observed the activation of JNK and phosphorylation of its direct target c-Jun along with inactivation of AKT and its direct target GSK in the course of programmed cell death. By means of small-molecule kinase inhibitors and transfection of cells with the genes of the components of these pathways, c-Jun and AKT, we confirm that JNK signalling pathway is proapoptotic, whereas AKT is antiapoptotic in daunorubicin-induced muscle cells. These findings could contribute to new approaches which will result in less toxicity and fewer side effects that are currently associated with the use of daunorubicin in cancer therapies.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.     

Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

It has been reported that miR-623 is deregulated in lung adenocarcinoma and inhibits tumor growth and invasion. However, it is unclear whether miR-623 has a role in the progression of hepatocellular carcinoma (HCC). Herein, we found that miR-623 was significantly downregulated in HCC, and that its expression was related to poor clinical outcomes of patients with HCC. Upregulation of miR-623 decreased cell proliferation, viability, migration, and invasion and further promoted apoptosis in 7721, Huh7, and Bel-7402 cells. Moreover, we also observed that miR-623 regulated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), Wnt/β-catenin, and extracellular regulated protein kinases/c-Jun N-terminal kinase (ERK/JNK) signaling pathways as well as the expression level of related proteins. Further, X-ray repair cross complementing 5 (XRCC5) was a direct target for miR-623, and the suppression of PI3K/Akt, Wnt/β-catenin, and ERK/JNK signaling pathways and cell proliferation and invasion abilities caused by miR-623 in HCC cells was significantly reversed by the upregulation of XRCC5. Collectively, our data suggested that miR-623 suppressed the progression of HCC by regulating the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by targeting XRCC5 in HCC in vitro, indicating that miR-623 may be a target for the therapy of HCC.     

Differential regulation of CIDEA and CIDEC expression by insulin via Akt1/2- and JNK2-dependent pathways in human adipocytes

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. Previously, we reported that CIDEA and CIDEC are differentially regulated by insulin and contribute separately to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. However, the upstream signals of CIDE proteins remain unclear. Here, we investigated the signaling molecules involved in insulin regulation of CIDEA and CIDEC expression. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 selectively inhibited insulin regulation of CIDEA and CIDEC expression, respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid droplet enlargement. Furthermore, insulin increased both Akt and JNK phosphorylation, which was abrogated by the PI3K inhibitors. These results suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it regulates expression of each protein via Akt1/2- and JNK2-dependent pathways, respectively, in human adipocytes.     

GINS2 facilitates epithelial-to-mesenchymal transition in non-small-cell lung cancer through modulating PI3K/Akt and MEK/ERK signaling

Non-small-cell lung cancer (NSCLC) is a cancer with high morbidity and mortality. We aimed to define the effect of Go-Ichi-Ni-San complex subuint 2 (GINS2) acting on NSCLC. The expressions of GINS2 in NSCLC tissues and cells were detected using real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry (IHC). The relationship between GINS2 expression and NSCLC prognosis or clinicopathologic features was analyzed through statistical analysis. The overexpressed or downexpressed plasmids of GINS2 were transfected into NSCLC cell lines, and then cell proliferation, invasion, and migration viability were, respectively, determined by Cell Counting Kit-8 assay, transwell, and wound healing assay. The epithelial–mesenchymal transition (EMT) was observed and the EMT-related proteins were measured using IHC and western blot. The function of GINS2 in vivo was assessed by mice model. The related proteins of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathways were evaluated using western blot. GINS2 expression was upregulated in NSCLC tissues and cell lines, and its high expression was correlated with the poor prognosis and several clinicopathologic features, such as TMN stages (tumor size, lymph node, and metastasis) and clinical stages. GINS2 enhanced NSCLC cell proliferation, migration, and invasion viability in vivo and in vitro. GINS2 also promoted NSCLC cells EMT. In addition, GINS2 could regulate phosphorylated proteins of PI3K p85, Akt, MEK, and ERK expressions, it revealed that GINS2 effected on PI3K/Akt and MEK/ERK pathways. GINS2 promoted cell proliferation, migration, invasion, and EMT via modulating PI3K/Akt and MEK/ERK signaling pathways. It might be a target in NSCLC treatment.     

Zinc chloride stimulates DNA synthesis of mouse embryonic stem cells: involvement of PI3K/Akt, MAPKs, and mTOR

Although zinc is one of the most important trace elements in the body, the mechanisms underlying zinc-induced cell proliferation have yet to be unraveled. Thus, we investigated the effect of zinc chloride (ZnCl(2)) on mouse embryonic stem (ES) cell proliferation and related signaling pathways. ZnCl(2) (40 microM) significantly increased [(3)H]-thymidine incorporation after 12 h of treatment. At moderate concentrations (> or =4 microM), ZnCl(2) increased cell cycle regulatory protein levels, [(3)H]-thymidine incorporation, and total cell numbers, but higher doses of ZnCl(2) (> or =200 microM) blocked this proliferative effect. ZnCl(2) induced the phosphorylation of Akt, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), p44/42 MAPKs, and mammalian target of rapamycin (mTOR) in a time-dependent manner. Pretreatment of LY 294002 (a PI3K inhibitor, 10(-6) M), wortmannin (a PI3K inhibitor, 10(-7) M), or an Akt inhibitor (10(-5) M), which inhibited the activation of JNK/SAPK and p44/42 MAPKs, blocked the ZnCl(2)-induced expression of cyclins and cyclin-dependent kinases (CDKs). Furthermore, pretreatment with PD 98059 (a p44/42 inhibitor, 10(-5) M) or SP 600125 (a JNK inhibitor, 10(-6) M) inhibited ZnCl(2)-induced activation of mTOR, p70S6K, and 4E-BP1. In addition, rapamycin (an mTOR inhibitor, 10(-8) M) blocked the ZnCl(2)-induced increase in [(3)H]-thymidine incorporation and cell cycle regulatory protein expression. In conclusion, ZnCl(2) stimulated ES cell proliferation through the PI3K/Akt, p44/42 MAPKs, JNK/SAPK, and mTOR signal pathways.     

Phosphoinositide 3‐kinases in cell migration

Cell migration is essential for many biological processes in animals and is a complex highly co‐ordinated process that involves cell polarization, actin‐driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3‐kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro‐migratory stimuli, and the diverse ways in which PI3K‐mediated signal transduction contributes to different aspects of cell migration.     

ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1

A possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2.     

Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA-NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K gamma gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kgamma. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.