Negative regulation of the SAPK/JNK signaling pathway by presenilin 1

Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid beta-protein. To understand intracellular signaling events downstream of PS1, we investigated in this study the action of PS1 on mitogen-activated protein kinase pathways. Overexpressed PS1 suppressed the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), failed to inhibit UV-stimulated SAPK/JNK. Furthermore, H(2)O(2-) or UV-stimulated SAPK activity was higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS(+/+) mice. MEF(PS1(-/-)) cells were more sensitive to the H(2)O(2)-induced apoptosis than MEF(PS1(+/+)) cells. Ectopic expression of PS1 in MEF(PS1(-/-)) cells suppressed H(2)O(2)-stimulated SAPK/JNK activity and apoptotic cell death. Together, our data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway.     

The neuronal SAPK/JNK pathway is altered in a murine model of hyperhomocysteinemia

Deficiency in cystathionine beta synthase (CBS) leads to high plasma homocysteine concentrations and causes hyperhomocysteinemia, a common risk factor for vascular disease, stroke and possibly neurodegenerative diseases. Various neuronal diseases have been associated with hyperhomocysteinemia, but the molecular mechanisms of homocysteine toxicity are unknown. We investigated the pathways involved in the pathological process, by analyzing differential gene expression in neuronal tissues. We used a combination of differential display and cDNA arrays to identify genes differentially expressed during hyperhomocysteinemia in brain of CBS-deficient mice. In this murine model of hyperhomocysteinemia, both plasma and brain homocysteine concentrations were high. Several genes were found to be differentially expressed in the brains of CBS-deficient mice, and the identities of some of these genes suggested that the SAPK/JNK pathway was altered in the brains of CBS-deficient mice. We therefore investigated the activation of proteins involved in the SAPK/JNK cascade. JNK and c-Jun were activated in the hippocampal neurones of CBS-deficient mice, suggesting that the SAPK/JNK pathway may play an important role in the development of neuronal defects associated with hyperhomocysteinemia.     

SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappaB-induced anti-apoptosis

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction ofsek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additionaltnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-Jun.     

HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.     

Activation of SAPK/JNK signaling by protein kinase Cdelta in response to DNA damage

The cellular response to genotoxic stress includes activation of protein kinase Cdelta (PKCdelta). The functional role of PKCdelta in the DNA damage response is unknown. The present studies demonstrate that PKCdelta is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1-beta-d-arabinofuranosylcytosine (araC) and other genotoxic agents. DNA damage-induced SAPK activation was attenuated by (i) treatment with rottlerin, (ii) expression of a kinase-inactive PKCdelta(K-R) mutant, and (iii) down-regulation of PKCdelta by small interfering RNA (siRNA). Coexpression studies demonstrate that PKCdelta activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1 --> MKK7 --> SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKCdelta, and in turn, MEKK1 is activated by a PKCdelta-dependent mechanism. These findings indicate that PKCdelta functions in the activation of SAPK through a Lyn --> PKCdelta --> MEKK1 --> MKK7 --> SAPK signaling cascade in response to DNA damage.     

JNK/SAPK activity contributes to TRAIL-induced apoptosis

We report here that JNK/SAPKs are activated by TRAIL in parallel to induction of apoptosis in human T and B cell lines. Death signaling as well as JNK/SAPK activation by TRAIL in these cells is FADD- and caspase-dependent since dominant-negative FADD or the caspase inhibitor zVAD prevented both, apoptosis and JNK/SAPK activity. JNK/SAPK activity in response to triggering of CD95 by an agonistic antibody (alphaAPO-1) was also diminished by dominant-negative FADD or zVAD. Correspondingly, a cell line resistant to alphaAPO-1-induced death exhibited crossresistance to TRAIL-induced apoptosis and did not upregulate JNK/SAPK activity in response to TRAIL or alphaAPO-1. Inhibition of JNK/SAPK activity, by stably transfecting cells with a dominant-negative JNKK-MKK4 construct, reduced apoptosis in response to TRAIL or alphaAPO-1. Therefore, activation of JNK/SAPKs by TRAIL or alphaAPO-1 occurs downstream of FADD and caspases and contributes to apoptosis in human lymphoid cell lines.     

ROS介导JNK信号通路的研究进展

c-JunN端激酶(JNK)通路是细胞感受外界环境变化的重要途径,与细胞增殖、分化、凋亡等生命过程息息相关.活性氧(ROS)具有很高的生物学活性,可作为第二信使参与到JNK信号通之中.ROS可通过ASK1、Src激酶、GSTπ、MLK3、RIP-TRAF2复合体、MKPs等信号蛋白活化JNK,也可以充当IKK/NF-κ B、ERK等信号通路与JNK信号通路交叉时话的桥梁.另外JNK有时可出现在ROS上游,可通过促进ROS产生或聚集而发挥生物学作用.本文将对近年来ROS介导JNK信号通路网络调控的研究进展作一综述.     

SUMO化信号途径对JNK信号通路活性的调控

c-Jun氨基末端激酶(the c-Jun N-terminal kinase,JNK)家族是促分裂原活化蛋白激酶(MAPK)超家族成员之一.JNK信号通路对细胞生长、分化和凋亡等生物学活动都有重要作用.而SUMO化是一种重要的生物学修饰,可以调节多种细胞生理活动.最近,黄海等在Development发表文章首次将SUMO化途径与JNK信号通路通过Hipk激酶联系起来,为进一步研究SUMO化的功能及其对JNK通路的调节建立了一个新的模型.     

Wortmannin-enhanced X-ray-induced apoptosis of human T-cell leukemia MOLT-4 cells possibly through the JNK/SAPK pathway

We demonstrated that enhancement of X-ray-induced apoptosis/rapid cell death by wortmannin accompanied by increased activation of JNK/SAPK in human leukemia MOLT-4 cells. Rapid cell death/apoptosis was determined either by the dye exclusion test or by the appearance of Annexin V-positive cells and cleaved PARP fragments. Enhancement was observed only at higher concentrations of wortmannin, i.e. 1 microM or more. At these high concentrations, both DNA-PK and ATM were inhibited. X-ray-induced phosphorylation of Ser 15 of p53/TP53, accumulation of both p53/TP53 and p21/WAF1/CDKN1A, and phosphorylation of XRCC4 were all suppressed. The enhancement of apoptosis/rapid cell death by wortmannin was prevented by addition of caspase inhibitors, Z-VAD-FMK or Ac-DEVD-CHO, or by transfection and overexpression of mouse Bcl2, which is known as an anti-apoptosis protein. The requirement for a high concentration of wortmannin, i.e. 1 microM or more, indicates that inhibition of both DNA-PK and ATM was necessary for the enhanced apoptosis/rapid cell death. Phosphorylation of AKT/PKB was completely suppressed at a much lower concentration, i.e. 0.1 microM wortmannin, where no enhancement of X-ray-induced apoptosis/rapid cell death was observed. On the other hand, X-ray-induced phosphorylation of JNK and its kinase activity as well as apoptosis/rapid cell death were all significantly enhanced only at high concentrations of wortmannin, i.e. 1 microM or more. Furthermore, the extent of enhancement of both JNK phosphorylation and of apoptosis/rapid cell death by wortmannin was less in Rh1a cells, which are ceramide- and radiation-resistant variant cells compared to the parental MOLT-4 cells. Therefore, activation of the JNK pathway was considered important for the enhancement of X-ray-induced apoptosis/rapid cell death of MOLT-4 cells by wortmannin, because of the requirement for a higher concentration of wortmannin than that required for inhibition of AKT phosphorylation. The suppression of the AKT-dependent pathway by wortmannin may have some underlying role in activating the JNK pathway toward the enhancement of cell death in the current system.     

LINC00707 promotes hepatocellular carcinoma progression through activating ERK/JNK/AKT pathway signaling pathway

Increasing evidence has demonstrated that abnormal expression of lncRNA is correlated with various malignant tumors, including hepatocellular carcinoma (HCC). Our current study was aimed to investigate the role of LINC00707 in HCC development. We observed that LINC00707 was upregulated in HCC cell lines compared with normal liver cell lines. Then, Hep3B cells and SNU449 cells were infected with LV-shLINC00707 and LV-LINC00707. LINC00707 silencing could greatly repress the proliferation and colony formation of HCC cells in vitro. On the contrary, overexpression of LINC00707 induced HCC cell proliferation and colony formation. In addition, HCC cell apoptosis was significantly enhanced and HCC cell cycle was blocked in G1 phase by LV-shLINC00707. Hep3B cells and SNU449 cell invasion capacity was restrained by the knockdown of LINC00707, whereas upregulation of LINC00707 exhibited an opposite phenomenon. Accumulating evidence has reported that ERK/JNK/AKT signaling is involved in multiple cancers, including HCC. Here, in our study, we identified that ERK/JNK/AKT signaling was dramatically restrained by silencing of LINC00707 while activated by LV-LINC00707 in HCC cells. Subsequently, an in vivo experiment was conducted, and it demonstrated that LINC00707 could modulate HCC development through activating ERK/JNK/AKT signaling. Taking the above results together, it was implied in our study that LINC00707 contributed to HCC progression through modulating the ERK/JNK/AKT pathway.     

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.     

Amyotrophic lateral sclerosis: a novel hypothesis involving a gained 'loss of function' in the JNK/SAPK pathway

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that mainly affects motor neurons. Despite intensive research efforts inspired by the mile-stone discovery linking the Cu/Zn superoxide dismutase 1 (SOD1) gene to a subset of familial cases, the mechanisms underlying disease pathogenesis are still largely unknown. Nonetheless, the recent finding of a second gene associated with familial form of the disease, ALS2, is likely to be of great help in elucidating the key pathways involved in motor neuron degeneration. Here, we provide evidence that the JNK/SAPK pathway plays a critical neuroprotective role in susceptible motor neurons in ALS. The involvement of the JNK/SAPK pathway integrates our knowledge about these two known genetic factors into a single pathogenic pathway involved in both sporadic and familial ALS.     

Amyotrophic lateral sclerosis: a novel hypothesis involving a gained 'loss of function' in the JNK/SAPK pathway

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that mainly affects motor neurons. Despite intensive research efforts inspired by the mile-stone discovery linking the Cu/Zn superoxide dismutase 1 (SOD1) gene to a subset of familial cases, the mechanisms underlying disease pathogenesis are still largely unknown. Nonetheless, the recent finding of a second gene associated with familial form of the disease, ALS2, is likely to be of great help in elucidating the key pathways involved in motor neuron degeneration. Here, we provide evidence that the JNK/SAPK pathway plays a critical neuroprotective role in susceptible motor neurons in ALS. The involvement of the JNK/SAPK pathway integrates our knowledge about these two known genetic factors into a single pathogenic pathway involved in both sporadic and familial ALS.     

Utidelone inhibits growth of colorectal cancer cells through ROS/JNK signaling pathway

Utidelone (UTD1), a novel microtubule stabilizing agent, is an epothilone B analogue which was produced by genetic engineering. UTD1 has exhibited broad antitumor activity in multiple solid tumors. However, its activity and mechanism in colorectal cancer (CRC) remain to be studied. In this study, UTD1 dramatically inhibited CRC cell proliferation (with 0.38 µg/ml, 0.77 µg/ml IC50 in RKO and HCT116, respectively) in vitro. Immunofluorescence staining showed that UTD1 induced the formation of microtubule bundling and asters in RKO cells. Flow cytometry analysis demonstrated that UTD1 induced cell cycle to arrest in G2/M phase, subsequent apoptosis. Significantly, UTD1 exhibited stronger effect on inducing apoptosis than paclitaxel and 5-FU, especially in HCT15 cells which is ABCB1 high-expression. UTD1 exposure cleaved caspase-3 and poly ADP-ribose polymerase (PARP), decreased mitochondrial membrane potential, released cytochrome c, increased the production of active oxygen and activated c-Jun N-terminal kinase (JNK), suggesting ROS/JNK pathway was involved in this process. Moreover, UTD1 inhibited tumor growth and was more effective and safer compared with paclitaxel and 5-FU in RKO xenograft in nude mice. Taken together, our findings first indicate that UDT1 inhibits tumor growth in CRC xenograft model and may be a promising agent for CRC treatment.Subject terms: Drug development, Preclinical research     

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.     

Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation

We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 phosphorylates the amino terminus of MEKK1 on serine 67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH(2)-terminal kinase/stress-activated protein kinases (JNK/SAPK). Serine 67 is constitutively phosphorylated in resting 293 cells, but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is negatively regulated by PAK through phosphorylation of serine 67.     

Integrin-dependent activation of the JNK signaling pathway by mechanical stress

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells.We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization.These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch.     

Roles of the JNK signaling pathway in Drosophila morphogenesis.

Epithelial cell differentiation and morphogenesis are crucial in many aspects of metazoan development. Recent genetic studies in Drosophila have revealed that the conserved Jun amino-terminal kinase (JNK) signaling pathway regulates epithelial morphogenesis during the process of embryonic dorsal closure and participates in the control of planar polarity in several tissues. Importantly, these studies have linked the JNK pathway to the decapentaplegic and Frizzled pathways in these processes, suggesting a high degree of integrative signaling during epithelial morphogenesis.