Dual Mkk4 and Mkk7 Gene Deletion in Adult Mouse Causes an Impairment of Hippocampal Immature Granule Cells.

(1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase involved in regulating physiological processes in the central nervous system. However, the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/-), Mkk4flox/flox, Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old mice induced the gene deletion (Actin-CreERT2 (Cre+/-), Mkk4∆/∆, Mkk7∆/∆ genotype), which was verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment position of immature hippocampal neurons together with alterations in their dendritic architecture pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: All these data supported that the MKK4/MKK7-JNK pathway has a role in adult neurogenic activity.

Novel tumor-suppressor function of KLF4 in pediatric T-cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in pediatric patients. Despite advances in the treatment of this disease, many children with T-cell ALL (T-ALL) die from disease relapse due to low responses to standard chemotherapy and the lack of a targeted therapy that selectively eradicates the chemoresistant leukemia-initiating cells (LICs) responsible for disease recurrence. We reported recently that the reprogramming factor Krüppel-like factor 4 (KLF4) has a tumor-suppressive function in children with T-ALL. KLF4 silencing by promoter deoxyribonucleic acid (DNA) methylation in patients with T-ALL leads to aberrant activation of the mitogen-activated protein kinase kinase MAP2K7 and the downstream c-Jun NH2-terminal kinase (JNK) pathway that controls the expansion of leukemia cells via c-Jun and activating transcription factor 2. This pathway can be inhibited with small molecules and therefore has the potential to eliminate LICs and eradicate disease in combination with standard therapy for patients with refractory and relapsed disease. The present review summarizes the role of the KLF4-MAP2K7 pathway in T-ALL pathogenesis and the function of JNK and MAP2K7 in carcinogenesis and therapy.

Heme oxygenase-1 protects spinal cord neurons from hydrogen peroxide-induced apoptosis via suppression of Cdc42/MLK3/MKK7/JNK3 signaling.

The mechanisms by which oxidative stress induces spinal cord neuron death has not been completely understood. Investigation on the molecular signal pathways involved in oxidative stress-mediated neuronal death is important for development of new therapeutics for oxidative stress-associated spinal cord disorders. In current study we examined the role of heme oxygenase-1 (HO-1) in the modulation of MLK3/MKK7/JNK3 signaling, which is a pro-apoptotic pathway, after treating primary spinal cord neurons with H2O2. We found that MLK3/MKK7/JNK3 signaling was substantially activated by H2O2 in a time-dependent manner, demonstrated by increase of activating phosphorylation of MLK3, MKK7 and JNK3. H2O2 also induced expression of HO-1. Transduction of neurons with HO-1-expressing adeno-associated virus before H2O2 treatment introduced expression of exogenous HO-1 in neurons. Exogenous HO-1 reduced phosphorylation of MLK3, MKK7 and JNK3. Consistent with its inhibitory effect on MLK3/MKK7/JNK3 signaling, exogenous HO-1 decreased H2O2-induced neuronal apoptosis and necrosis. Furthermore, we found that exogenous HO-1 inhibited expression of Cdc42, which is crucial for MLK3 activation. In addition, HO-1-induced down-regulation of MLK3/MKK7/JNK3 signaling might be related to up-regulation of microRNA-137 (mir-137). A mir-137 inhibitor alleviated the inhibitory effect of HO-1 on JNK3 activation. This inhibitor also increased neuronal death even when exogenous HO-1 was expressed. Therefore, our study suggests a novel mechanism by which HO-1 exerted its neuroprotective efficacy on oxidative stress.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

OBJECTIVE: Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. METHODS: We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. RESULTS: ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor ß-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. CONCLUSION: The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL-1 stimulation because of LRP-1 shedding.

Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity.

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four "stomatal" MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.

A non-redundant role for Drosophila Mkk4 and hemipterous/Mkk7 in TAK1-mediated activation of JNK.

BACKGROUND: The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1. METHODOLOGY/PRINCIPAL FINDINGS: In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation. CONCLUSIONS/SIGNIFICANCE: In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.

Distinct contributions of JNK and p38 to chromium cytotoxicity and inhibition of murine embryonic stem cell differentiation.

BACKGROUND: Potassium dichromate [Cr(VI)] is a widespread environmental toxicant responsible for increased risk of several human diseases. Cr(VI) exposure leads to activation of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK)1/2, p38, and extracellular-signal regulated kinase (ERK)1/2. OBJECTIVES: We evaluated the contribution of MAPKs to Cr(VI) toxicity. METHODS: Phosphorylation of MAPKs and their downstream effectors was evaluated by Western immunoblotting; reactive oxygen species were measured by DCFDA (5',6'-chloromethyl-2'-7'-dichlorofluorescin diacetate) labeling and flow cytometry, and glutathione and glutathione disulfide levels were determined by monochrome graphic spectroflurometer. Cytotoxicity was assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation. Embryoid body (EB) differentiation was evaluated by contracting cardiomyocyte formation, and real-time polymerase chain reaction (RT-PCR) was used for cardiomyocyte-specific and stem-cell-specific gene expression. RESULTS: Acute treatment of mouse embryonic stem (ES) cells with 50 microM Cr(VI) induced the rapid phosphorylation of JNK, p38, and ERK and their respective downstream transcription factors, c-JUN, activating transcription factor-2, and ELK1. MAPK activation and cytotoxicity induction were partially blocked by pretreatment with the antioxidant N-acetyl cysteine. Ablation of the upstream MAP kinase kinase (MAP2K7) in ES cells prevented JNK activation, whereas ablation of MAP2K4 prevented both JNK and p38 activation. Using specific MAPK inhibitors and MAP2K4- and MAP2K7-deficient ES cells, we showed that JNK reduced acute Cr(VI) cytotoxicity, p38 potentiated it, and ERK had no effect. At low submicromolar concentrations, Cr(VI) caused MAP2K4/7-dependent JNK activation and MAP2K4-dependent p38 activation and strongly inhibited contracting cardiomyocyte development in wild-type ES cells, but much less so in Map2k7((-/-)) cells. CONCLUSION: Each MAPK distinctly contributes to chromium toxicity. Whereas JNK prevents and p38 promotes acute cytotoxicity, JNK contributes to optimal inhibition of ES cell differentiation by chromium.

Metabolic stress signaling mediated by mixed-lineage kinases.

Saturated free fatty acid (FFA) is a major source of metabolic stress that activates the c-Jun NH(2)-terminal kinase (JNK). This FFA-stimulated JNK pathway is relevant to hallmarks of metabolic syndrome, including insulin resistance. Here we used gene ablation studies in mice to demonstrate a central role for mixed-lineage protein kinases (MLK) in this signaling pathway. Saturated FFA causes protein kinase C (PKC)-dependent activation of MLK3 that subsequently causes increased JNK activity by a mechanism that requires the MAP kinase kinases MKK4 and MKK7. Loss of PKC, MLK3, MKK4, or MKK7 expression prevents FFA-stimulated JNK activation. Together, these data establish a signaling pathway that mediates effects of metabolic stress on insulin resistance.

Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling.

MEKK2 and MEK5 encode Phox/Bem1p (PB1) domains that heterodimerize with one another. MEKK2, MEK5, and extracellular signal-related kinase 5 (ERK5) form a ternary complex through interactions involving the MEKK2 and MEK5 PB1 domains and a 34-amino-acid C-terminal extension of the MEK5 PB1 domain. This C-terminal extension encodes an ERK5 docking site required for MEK5 activation of ERK5. The PB1 domains bind in a front-to-back arrangement, with a cluster of basic amino acids in the front of the MEKK2 PB1 domain binding to the back-end acidic clusters of the MEK5 PB1 domain. The C-terminal moiety, including the acidic cluster of the MEKK2 PB1 domain, is not required for MEK5 binding and binds MKK7. Quiescent MEKK2 preferentially binds MEK5, and MEKK2 activation results in ERK5 activation. Activated MEKK2 binds and activates MKK7, leading to JNK activation. The findings define how the MEKK2 and MEK5 PB1 domains are uniquely used for differential binding of two mitogen-activated protein kinase kinases, MEK5 and MKK7, for the coordinated control of ERK5 and c-Jun N-terminal kinase activation.

Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia.

Our previous study indicates that global ischemia facilitates the assembly of the GluR6.PSD-95.MLK3 signaling module, which in turn activated MLK3, leading to exacerbated ischemic neuron death. In addition, JIP1, functioning as a scaffold protein, could couple MLK3-MKK7-JNK to form a specific signaling module and facilitate the activation of the JNK signal pathway. However, the organization, regulation, and function between the two signaling modules and the effects they have on MLK3 activation remain incompletely understood. Here, we show that JIP1 maintains MLK3 in an inactive and monomeric state; once activated, MLK3 binds to PSD-95 and then dimerizes and autophosphorylates. In addition, a GluR6 C-terminus-containing peptide (Tat-GluR6-9c) and antisense oligonucleotides (AS-ODNs) against PSD-95 inhibit the integration of PSD-95 and MLK3 and the dimerization of MLK3, facilitate the interaction of JIP1 and MLK3, and, consequently, perform neuroprotection on neuron death. However, AS-ODNs against JIP1 play a negative role compared to that mentioned above. The findings show that the crosstalk occurs between PSD-95 and the JIP1-mediated signaling module, which may be involved in brain ischemic injury and contribute to the regulation of MLK3 activation. Thus, specific blockade of PSD-95-MLK3 coupling may reduce the extent of ischemia-reperfusion-induced neuronal cell death.

Physiological roles of MKK4 and MKK7: insights from animal models.

c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of numerous physiological processes during development and in response to stress. Its activity is increased upon phosphorylation by the MAPK kinases, MKK4 and MKK7. Similar to the early embryonic death of mice caused by the targeted deletion of the jnk genes, mice lacking mkk4 or mkk7 die before birth. The inability of MKK4 and MKK7 to compensate for each other's functions in vivo is consistent with their synergistic effect in mediating JNK activation. However, the phenotypic analysis of the mutant mouse embryos indicates that MKK4 and MKK7 have specific roles that may be due to their selective regulation by extracellular stimuli and their distinct tissue distribution. MKK4 and MKK7 also have different biochemical properties. For example, whereas MKK4 can activate p38 MAPK, MKK7 functions as a specific activator of JNK. Here we summarize the studies that have shed light on the mechanism of activation of MKK4 and MKK7 and on their physiological functions.

Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins.

Different scaffold proteins play distinct roles in various signaling pathways by recruiting different downstream molecules. Here, using MKK4(-/-) and MKK4(-/-)/7(-/-) murine embryonic fibroblast cells, we examined differential employment of MKK4 and MKK7 by scaffold proteins Axin, Dvl, and Epstein-Barr virus latent membrane protein-1 (LMP-1) in mediating JNK activation. We present evidence that Axin depends mainly on MKK7 for activation of JNK, while Dvl depends almost equally on MKK4 and MKK7 for JNK activation, In contrast, LMP-1-induced JNK activation is primarily dependent on MKK4. Our results demonstrate that Axin, Dvl, and LMP-1 differentially utilize MKK4 and MKK7 for JNK activation.

Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6).

UNLABELLED: This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-kappaB signaling. INTRODUCTION: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. MATERIALS AND METHODS: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-kappaB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IkappaB kinase 2 (IKK(DN)) gene and mitogen-activated protein kinase kinase 7 (MKK7(DN)) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. RESULTS AND CONCLUSION: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-kappaB pathway by IKK(DN) overexpression, but not that of the JNK pathway by MKK7(DN) overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-kappaB-mediated downregulation is essential for efficient osteoclast differentiation.