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This article explores the molecular mechanisms underlying early brain injury (EBI) following subarachnoid hemorrhage (SAH). The researchers found that inhibiting histone deacetylase 4 (HDAC4) decreased neuronal apoptosis and brain injury by suppressing the transcription of MKK7, an upstream kinase of the JNK/c-Jun pathway. The study suggests that targeting HDAC4 could be a potential strategy for preventing SAH-induced EBI. The article provides insights into the role of HDAC4 in regulating neuronal apoptosis and the JNK/c-Jun signaling pathway, offering potential therapeutic implications for neurodegenerative diseases and acute brain injuries. [Extracted from the article]

SUMMARY: Plant diseases, which seriously damage crop production, are in most cases caused by fungal pathogens. In this study, we found that the Raf‐like MAPKKKs STY8 (SERINE/THREONINE/TYROSINE KINASE 8), STY17, and STY46 negatively regulate resistance to the fungal pathogen Botrytis cinerea through jasmonate response in Arabidopsis. Moreover, STY8/STY17/STY46 homologs negatively contribute to chitin signaling. We further identified MKK7 as the MAPKK component interacting with STY8/STY17/STY46 homologs. MKK7 positively contributes to resistance to B. cinerea and chitin signaling. Furthermore, we found that STY8/STY17/STY46 homologs negatively affect the accumulation of MKK7, in accordance with the opposite roles of MKK7 and STY8/STY17/STY46 homologs in defense against B. cinerea. These results provide new insights into the mechanisms precisely regulating plant immunity via Raf‐like MAPKKKs. Significance Statement: We found that STY8/STY17/STY46 homologs negatively regulate plant resistance through modulating the MKK7 pathway. [ABSTRACT FROM AUTHOR]

Purpose: Mitogen-activated protein kinases (MAPK), specifically the c-Jun N-terminal kinase (JNK)–MAPK subfamily, play a crucial role in the development of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of JNK1/2 and their upstream regulators, MKK4/7, in HCC carcinogenesis remain unclear. Methods: In this study, we performed differential expression analysis of JNK–MAPK components at both the transcriptome and protein levels using TCGA and HPA databases. We utilized Kaplan–Meier survival plots and receiver operating characteristic (ROC) curve analysis to evaluate the prognostic performance of a risk scoring model based on these components in the TCGA-HCC cohort. Additionally, we conducted immunoblotting, apoptosis analysis with FACS and soft agar assays to investigate the response of JNK–MAPK pathway components to various death stimuli (TRAIL, TNF-α, anisomycin, and etoposide) in HCC cell lines. Results: JNK1/2 and MKK7 levels were significantly upregulated in HCC samples compared to paracarcinoma tissues, whereas MKK4 was downregulated. ROC analyses suggested that JNK2 and MKK7 may serve as suitable diagnostic genes for HCC, and high JNK2 expression correlated with significantly poorer overall survival. Knockdown of JNK1 enhanced TRAIL-induced apoptosis in hepatoma cells, while JNK2 knockdown reduced TNF-α/cycloheximide (CHX)—and anisomycin-induced apoptosis. Neither JNK1 nor JNK2 knockdown affected etoposide-induced apoptosis. Furthermore, MKK7 knockdown augmented TNF-α/CHX- and TRAIL-induced apoptosis and inhibited colony formation in hepatoma cells. Conclusion: Targeting MKK7, rather than JNK1/2 or MKK4, may be a promising therapeutic strategy to inhibit the JNK–MAPK pathway in HCC therapy. [ABSTRACT FROM AUTHOR]

Objective: To investigate the mechanism of action of tetrahydroxy stilbene glycosides(TSG) in ameliorating neuronal damage in Alzheimer's disease rats by regulating MKK7 and JNK kinases. Methods: A total of 24-month-old 42 SD rats were randomly selected for the experiment in 7 groups: normal group, sham-operated group, model group, positive drug group, low, medium and high dose TSG group at 0.033 g/kg, 0.1 g/kg, 0.3 g/kg. The Model Group and the TSG groups were established by stereotaxic Aß25-35 solution. After 28 days, the model rats were selected by passive avoidance test. After screening, each dosage group of TSG and positive drug group was given intragastrically according to the corresponding dosage, and the experiment was carried out after 28 days. The pathological changes of hippocampal CA1 region were observed by tissue staining, and the amount of MKK7 and JNK proteins and the expression content of MKK7 and JNK mRNA by histochemical method of protein, and qRTPCR assay. Results: (1) He staining observation: Compared with the normal group and the sham-operated group, the number of nerve cells in the model group decreased and arranged irregularly, the cell membrane shrank, and the nucleus deformed and dissolved. The number of neurons in the positive drug group and TSG Group also increased significantly, the order is also relatively well. (2) From the results of the Tunel staining experiments:the positive apoptotic cells in the model group were higher than control group and sham-operated group, positive drug group and TSG drugs group was significantly smaller than that in the model group (P < 0.05). (3) Compared with the control group and the Virtual Operation Group, the MKK7 and JNK protein concentrations in the brain of the model group were increased(P<0.05) by data analysis of immunohistochemistry: Compared with the model group, the protein expression of positive drug and TSG each dose group were reduced (P<0.05). (4)The results of QRTPCR data showed that the levels of MKK7 and JNK mRNA in the brain tissue of the model group were increased compared with the normal group and sham-operated groups(P<0.05). Conclusion: Stilbene glycoside has a certain effect on neuronal injury and repair which may be related to the changes of mRNA transcription and protein expression of MKK7 and JNK kinases. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression of phospho-c-Jun and MKK7 in gliomas and their correlation. Methods We collected and analyzed retrospectively 92 cases of gliomas, including 15 cases of diffuse astrocytoma, 5 cases of oligodendroglioma, 11 cases of anaplastic astrocytoma, 8 cases of anaplastic oligodendroglioma 53 cases of glioblastoma and 25 normal brain tissues adjacent to glioblastoma. The expression of c-Jun, phospho-c-Jun and MKK7 were detected by immunohistochemical staining. Glioma U87 cell line cultured in vitro was transfected with MKK4-siRNA, MKK7-siRNA and control siRNA for 48h, respectively. Western blot was performed to test the expression of c-Jun, phospho-c-Jun and MKK7. Results The expression of p-c-Jun and MKK7 in glioblastoma were significantly higher than those in other glioma and normal brain tissues adjacent to glioblastomas(P=0.000, P=0.000). The expression of MKK7 and phospho-c-Jun were positively correlated with WHO grading of gliomas (r＝0.494, P＝0.000; r=0.606, P=0.000). There was positive correlation between the expression of MKK7 and p-c-Jun (r=0.387, P=0.000). The knockdown of MKK7 suppressed c-Jun activities. Conclusion MKK7 promotes the development of glioblastoma by regulating the activity of c-Jun.

c‐Jun N‐terminal kinases (JNKs) are constitutively activated in mammalian brains and are indispensable for their development and neural functions. MKK7 is an upstream activator of all JNKs. However, whether the common JNK signaling pathway regulates the brain's control of social behavior remains unclear. Here, we show that female mice in which Mkk7 is deleted specifically in mature neurons (Mkk7flox/floxSyn‐Cre mice) give birth to a normal number of pups but fail to raise them due to a defect in pup retrieval. To explore the mechanism underlying this abnormality, we performed comprehensive behavioral tests. Mkk7flox/floxSyn‐Cre mice showed normal locomotor functions and cognitive ability but exhibited depression‐like behavior. cDNA microarray analysis of mutant brain revealed an altered gene expression pattern. Quantitative RT‐PCR analysis demonstrated that mRNA expression levels of genes related to neural signaling pathways and a calcium channel were significantly different from controls. In addition, loss of neural MKK7 had unexpected regulatory effects on gene expression patterns in oligodendrocytes. These findings indicate that MKK7 has an important role in regulating the gene expression patterns responsible for promoting normal social behavior and staving off depression. [ABSTRACT FROM AUTHOR]

The c-Jun N-terminal kinase (JNK)/c-Jun cascade-dependent neuronal apoptosis has been identified as a central element for early brain injury (EBI) following subarachnoid hemorrhage (SAH), but the molecular mechanisms underlying this process are still thoroughly undefined to date. In this study, we found that pan-histone deacetylase (HDAC) inhibition by TSA, SAHA, VPA, and M344 led to a remarkable decrease in the phosphorylation of JNK and c-Jun, concomitant with a significant abrogation of apoptosis caused by potassium deprivation in cultured cerebellar granule neurons (CGNs). Further investigation showed that these effects resulted from HDAC inhibition-induced transcriptional suppression of MKK7, a well-known upstream kinase of JNK. Using small interference RNAs (siRNAs) to silence the respective HDAC members, HDAC4 was screened to be required for MKK7 transcription and JNK/c-Jun activation. LMK235, a specific HDAC4 inhibitor, dose-dependently suppressed MKK7 transcription and JNK/c-Jun activity. Functionally, HDAC4 inhibition via knockdown or LMK235 significantly rescued CGN apoptosis induced by potassium deprivation. Moreover, administration of LMK235 remarkably ameliorated the EBI process in SAH rats, associated with an obvious reduction in MKK7 transcription, JNK/c-Jun activity, and neuronal apoptosis. Collectively, the findings provide new insights into the molecular mechanism of neuronal apoptosis regarding HDAC4 in the selective regulation of MKK7 transcription and JNK/c-Jun activity. HDAC4 inhibition could be a potential alternative to prevent MKK7/JNK/c-Jun axis-mediated nervous disorders, including SAH-caused EBI.

Histone deacetylase 6 (HDAC6) activity contributes to the malignant proliferation, invasion, and migration of glioma cells (GCs), but the molecular mechanisms underlying the processes remains elusive. Here, we reported that HDAC6 inhibition by Ricolinostat (ACY‐1215) or CAY10603 led to a remarkable decrease in the phosphorylation of c‐Jun N‐terminal kinase (JNK) and c‐Jun, which preceded its suppressive effects on glioma cell growth. Further investigation showed that these effects resulted from HDAC6 inhibitor‐induced suppression of MAPK kinase 7 (MKK7), which was identified to be critical for JNK activation and exerts the oncogenic roles in GCs. Selectively silencing HDAC6 by siRNAs had the same responses, whereas transient transfections expressing HDAC6 promoted MKK7 expression. Interestingly, by performing Q‐PCR, HDAC6 inhibition did not cause a down‐regulation of MKK7 mRNA level, whereas the suppressive effects on MKK7 protein can be efficiently blocked by the proteasomal inhibitor MG132. As a further test, elevating MKK7‐JNK activity was sufficient to rescue HDAC6 inhibitor‐mediated‐suppressive effects on c‐Jun activation and the malignant features. The suppression of both MKK7 expression and JNK/c‐Jun activities was involved in the tumor‐growth inhibitory effects induced by CAY10603 in U87‐xenograft mice. Collectively, our findings provide new insights into the molecular mechanism of glioma malignancy regarding HDAC6 in the selective regulation of MKK7 expression and JNK/c‐Jun activity. MKK7 protein stability critically depends on HDAC6 activity, and inhibition of HDAC6 probably presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c‐Jun axis in GCs. [ABSTRACT FROM AUTHOR]

JNK activity has been implicated in the malignant proliferation, invasion and drug‐resistance of glioma cells (GCs), but the molecular mechanisms underlying JNK activation are currently unknown. Here, we reported that MKK7, not MKK4, directly activates JNK in GCs and exerts oncogenic effects on tumor formation. Notably, MKK7 expression in glioma tissues was closely correlated with the grade of the glioma and JNK/c‐Jun activation. Mechanistically, MKK7 transcription critically depends on the complexes formed by HDAC4 and the transcriptional factors SP1 and Krüppel‐like factor‐5 (KLF5), wherein HDAC4 directly deacetylates both SP1 and KLF5 and synergistically upregulates MKK7 transcription through two SP1 sites located on its promoter. In contrast, the increases in acetylated‐SP1 and acetylated‐KLF5 after HDAC4 inhibition switched to transcriptionally suppress MKK7. Selective inhibition of HDAC4 by LMK235, siRNAs or blockage of SP1 and KLF5 by the ectopic dominant‐negative SP1 greatly reduced the malignant capacity of GCs. Furthermore, suppression of both MKK7 expression and JNK/c‐Jun activities was involved in the tumor‐growth inhibitory effects induced by LMK235 in U87‐xenograft mice. Interestingly, HDAC4 is highly expressed in glioma tissues, and the rate of HDAC4 nuclear import is closely correlated with glioma grade, as well as with MKK7 expression. Collectively, these findings demonstrated that highly expressed MKK7 contributes to JNK/c‐Jun signaling‐mediated glioma formation. MKK7 transcription, regulated by SP1 and KLF5, critically depends on HDAC4 activity, and inhibition of HDAC4 presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c‐Jun signaling in GCs. What's new? Given their poor prognosis, new glioma therapies are urgently needed. The protein kinase JNK has been implicated in the proliferation, invasion and multiple‐drug resistance of glioma cells. But how is JNK activated? This study revealed that an enzyme called MKK7 directly activates the JNK/c‐Jun pathway in these cells. The authors were also able to identify the complex mechanism by which this occurs. Their results indicate that inhibition of the enzyme HDAC4 may offer a new therapeutic strategy for suppressing the oncogenic roles of MKK7/JNK/c‐Jun signaling in glioma cells. [ABSTRACT FROM AUTHOR]

The c-Jun N-terminal kinase (JNK)/c-Jun cascade-dependent neuronal apoptosis has been identified as a central element for early brain injury (EBI) following subarachnoid hemorrhage (SAH), but the molecular mechanisms underlying this process are still thoroughly undefined to date. In this study, we found that pan-histone deacetylase (HDAC) inhibition by TSA, SAHA, VPA, and M344 led to a remarkable decrease in the phosphorylation of JNK and c-Jun, concomitant with a significant abrogation of apoptosis caused by potassium deprivation in cultured cerebellar granule neurons (CGNs). Further investigation showed that these effects resulted from HDAC inhibition-induced transcriptional suppression of MKK7, a well-known upstream kinase of JNK. Using small interference RNAs (siRNAs) to silence the respective HDAC members, HDAC4 was screened to be required for MKK7 transcription and JNK/c-Jun activation. LMK235, a specific HDAC4 inhibitor, dose-dependently suppressed MKK7 transcription and JNK/c-Jun activity. Functionally, HDAC4 inhibition via knockdown or LMK235 significantly rescued CGN apoptosis induced by potassium deprivation. Moreover, administration of LMK235 remarkably ameliorated the EBI process in SAH rats, associated with an obvious reduction in MKK7 transcription, JNK/c-Jun activity, and neuronal apoptosis. Collectively, the findings provide new insights into the molecular mechanism of neuronal apoptosis regarding HDAC4 in the selective regulation of MKK7 transcription and JNK/c-Jun activity. HDAC4 inhibition could be a potential alternative to prevent MKK7/JNK/c-Jun axis-mediated nervous disorders, including SAH-caused EBI. [ABSTRACT FROM AUTHOR]

(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.

One of the mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal protein kinase (JNK) plays an important role in regulating cell fate, such as proliferation, differentiation, development, transformation, and apoptosis. Its activity is induced through the interaction of MAPK kinase kinases (MAP3Ks), MAPK kinases (MAP2Ks), and various scaffolding proteins. Because of the importance of the JNK cascade to intracellular bioactivity, many studies have been conducted to reveal its precise intracellular functions and mechanisms, but its regulatory mechanisms remain elusive. In this review, we discuss the molecular characterization, activation process, and physiological functions of mitogen-activated protein kinase kinase 7 (MKK7), the MAP2K that most specifically controls the activity of JNK. Understanding the role of MKK7/JNK signaling in physiological conditions could spark new hypotheses for targeted anticancer therapies. [ABSTRACT FROM AUTHOR]

Repetitive transcranial magnetic stimulation (rTMS) is a technique protecting neurons against diverse neurodegenerative disorders by delivering magnetic stimuli into the brain through the intact scalp. In the current study, the protection effect of rTMS on Parkinson's disease (PD) and the associated mechanism driving the treatment were explored. The PD symptoms were induced using 6-OHDA in mice, and the effect of rTMS of two frequencies (1 Hz and 10 Hz) on the cognitive behaviors and neuron viability was detected. Afterwards, the level of Aβ1–42 and activity of MKK7-ERK-Fos-APP axis under the administration of rTMS were recorded as well. The intracranial injection of 6-OHDA impaired the cognitive behaviors of the mice in the test of Morris water maze as well as reducing the viability and number of neurons in PD mice. After the treatment of rTMS of both frequencies, the cognitive function of mice was improved and the neuron viability and number were restored in mice brain tissues. The administration of rTMS also increased the cerebrospinal fluid (CSF) level of Aβ1–42 in PD mice, which was accompanied by the suppressed levels of p-MKK7, p-ERK1/2, p–c-Fos, and APP. Moreover, the effect of rTMS on mice nerve system was all exerted in a frequency-dependent manner. In conclusion, the findings outlined in the current study affirmed the protection effect of rTMS against PD. The anti-PD function of rTMS was associated with the suppression of MKK7-ERK-Fos-APP axis, which subsequently resulted in the increased CSF Aβ1–42 level and decreased brain Aβ1–42 level. [ABSTRACT FROM AUTHOR]

GADD45β/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45β/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45β/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

JNK, a member of the mitogen activated protein kinases (MAPKs) superfamily, plays a key role in cell death in many neurological disorders, but systemic inhibition of JNK has detrimental side effects. JNK can be regulated by two direct upstream kinases: MAPK kinase 4 (MKK4) and MAPK kinase 7 (MKK7). Here, we investigated the effect of GADD45β-I, a recently designed cell-permeable inhibitor peptide for MKK7, on endoplasmic reticulum (ER) stress-induced cytotoxicity in neuronal HT22 cells. We found that treatment with the ER stress inducer tunicamycin (TM) increased the phosphorylation of JNK and MKK7 in HT22 cells, which was nullified by GADD45β-I. GADD45β-I significantly attenuated TM-induced toxicity via inhibiting apoptotic cell death, as evidenced by decreased number of TUNEL-positive cells and reduced caspase-3 activity. GADD45β-I treatment also decreased expression of ER stress associated pro-apoptotic proteins and prevented morphological changes of the ER after TM exposure. In addition, inhibition of mitochondrial oxidative stress and preservation of intracellular ATP levels were observed in GADD45β-I-treated cells. The experiments using siRNA transfection and Topflash reporter assay revealed a possible involvement of Wnt/β-catenin pathway in GADD45β-I-induced protection in HT22 cells. In summary, our results demonstrated that GADD45β-I exerted protective effects against TM-induced cytotoxicity via regulating JNK-Wnt pathway. Targeting MKK7 could represent a new therapeutic strategy for the treatment of neurological diseases where ER stress associated neuronal injury are involved. [ABSTRACT FROM AUTHOR]

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues. [ABSTRACT FROM AUTHOR]

Intestinal ischemia reperfusion (I/R) may cause inflammation-, oxidative stress-, and apoptosis-related tissue injuries and facilitate bacterial infection, leading to multiple organ failure. Myricetin, a flavonoid, is found to have diverse biological effects including anti-inflammatory, anti-oxidative, and anti-bacterial effects. Based on our pre-experiment, we proposed that myricetin pretreatment (25, 50 mg/kg) could ameliorate intestinal I/R injury and myricetin-induced modulation on MKK7/JNK signal pathway might play a key role in the amelioration. The present study was designed to verify the proposal by using both rat intestinal I/R model in vivo and hypoxia/reoxygenation (H/R)-injured intestinal epithelial cell line (IEC-6 cells) model in vitro. The results confirmed our proposal. Myricetin selectively ameliorated I/R- and H/R-induced injuries in vivo and in vitro respectively without significantly affecting the corresponding normal controls. Myricetin significantly alleviated I/R-induced rat intestinal injury by reducing the generation of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 and by reducing MPO activity. Myricetin significantly reduced oxidative stress through decreasing MDA level and increasing the levels of SOD and GSH in the intestinal tissues compared with I/R control rats. Myricetin significantly decreased apoptosis by selectively down-regulating the expression of p-MKK7 and p-JNK without affecting MKK7 and JNK, inhibiting Bax, caspase-3 protein expression, and up-regulating Bcl-2 protein expression in I/R-injured jejunum of rats. In vitro study indicated that MKK7 siRNA transfection significantly decreased both MKK7 and p-MKK7 and other apoptosis-related proteins, partially simulating myricetin-induced anti-apoptotic effects. MKK7 siRNA transfection + myricetin could not further decrease MKK7, p-MKK7, and other apoptosis-related proteins, suggesting that inhibition of MKK7/JNK pathway plays a key role in myricetin-induced protection against intestinal I/R. MKK7 overexpression by cDNA transfection abrogated myricetin-reduced apoptosis-related protein expression, confirming that the MKK7/JNK signal pathway is the key target for myricetin-induced amelioration. The present study indicated that pretreatment of myricetin induced selective protection against intestinal I/R injury without significantly affecting corresponding normal controls and p-MKK7 was the key target, suggesting that myricetin is worth further translational studies. [ABSTRACT FROM AUTHOR]

Growth arrest and DNA damage-inducible 45 (GADD45) family genes encode related proteins, including GADD45α, GADD45β, and GADD45γ. In HeLa cells, expression of GADD45 members is differentially regulated under a variety of environmental conditions, but thermal and genotoxic stresses induce the expression of all genes. The heat shock response of GADD45β is mediated by the heat shock transcription factor 1 (HSF1), and GADD45β is necessary for heat stress survival. Heat and genotoxic stress-induced activation of c-Jun N-terminal kinase (JNK) is suppressed by the expression of GADD45 proteins. GADD45 proteins bind the JNK kinase mitogen-activated protein kinase kinase 7 (MKK7) and inhibit its activity, even under normal physiological conditions. Our findings indicate that GADD45 essentially suppresses the MKK7-JNK pathway and suggest that differentially expressed GADD45 family members fine-tune stress-inducible JNK activity. [ABSTRACT FROM AUTHOR]

c-Jun-N-terminal kinase (JNK) activity plays a critical role in modulating cell death, which depends on the level and duration of JNK activation. The kinase cascade from MAPkinase kinase kinase (MAP3K) to MAPkinase kinase (MAP2K) to MAPKinase (MAPK) can be regulated by a number of direct and indirect post-transcriptional modifications, including acetylation, ubiquitination, phosphorylation, and their reversals. Recently, a JNK-mitochondrial SH3-domain binding protein 5 (SH3BP5/SAB)-ROS activation loop has been elucidated, which is required to sustain JNK activity. Importantly, the level of SAB expression in the outer membrane of mitochondria is a major determinant of the set-point for sustained JNK activation. SAB is a docking protein and substrate for JNK, leading to an intramitochondrial signal transduction pathway, which impairs electron transport and promotes reactive oxygen species (ROS) release to sustain the MAPK cascade.

Rationale: Members of the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein (MAP) kinases, and the upstream kinase MKK7, have all been strongly linked with synaptic plasticity and with the development of the neocortex. However, the impact of disruption of this pathway on cognitive function is unclear. Objective: In the current study, we test the hypothesis that reduced MKK7 expression is sufficient to cause cognitive impairment. Methods: Attentional function in mice haploinsufficient for Map2k7 ( Map2k7 mice) was investigated using the five-choice serial reaction time task (5-CSRTT). Results: Once stable performance had been achieved, Map2k7 mice showed a distinctive attentional deficit, in the form of an increased number of missed responses, accompanied by a more pronounced decrement in performance over time and elevated intra-individual reaction time variability. When performance was reassessed after administration of minocycline-a tetracycline antibiotic currently showing promise for the improvement of attentional deficits in patients with schizophrenia-signs of improvement in attentional performance were detected. Conclusions: Overall, Map2k7 haploinsufficiency causes a distinctive pattern of cognitive impairment strongly suggestive of an inability to sustain attention, in accordance with those seen in psychiatric patients carrying out similar tasks. This may be important for understanding the mechanisms of cognitive dysfunction in clinical populations and highlights the possibility of treating some of these deficits with minocycline. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase 7 (MKK7) is one of the major stress-activated protein kinase (SAPK)-activating kinases in response to environmental or physiological stimuli. Here a MKK7 named as Ec-MKK7 was identified from orange-spotted grouper, Epinephelus coioides . The full-length cDNA of Ec-MKK7 was 1853 bp, with an open reading frame (ORF) of 1272 bp encoding a putative protein of 423 amino acids. A characteristic S-K-A-K-T motif was contained in the domain of dual-specificity protein kinase, mitogen-activated protein kinase kinase 7 (PKc_MKK7). Intracellular localization showed that Ec-MKK7 was localized in both the cytoplasm and the nucleus of grouper spleen (GS) and/or grouper brain (EAGB) cells. Moreover, Ec-MKK7 was universally expressed in all examined tissues and showed expression modulation to challenges of lipopolysacchride (LPS), Singapore grouper iridovirus (SGIV) and polyriboinosinic polyribocytidylic acid (poly I:C) in vivo . A gene targeting strategy over-expressing Ec-MKK7 was performed to examine the activities of MKK7 to viral infection in vitro . Our data showed that Ec-MKK7 was involved in the evasion and replication of SGIV but played an antiviral role to the infection of nervous necrosis virus (NNV). All results demonstrated that Ec-MKK7 could play important roles in grouper innate immunity and show distinct functions on virus infection. [ABSTRACT FROM AUTHOR]

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then upregulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)- HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7- JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway. [ABSTRACT FROM AUTHOR]

Reprogramming of somatic cells to the phenotypic state termed 'induced pluripotency' is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well-established molecular barriers in human somatic cell induced reprogramming. S tem C ells 2016;34:1198-1212 [ABSTRACT FROM AUTHOR]

Objectives: Krüppel-like factor 5 (KLF5) modulates multiple cell processes in different cancers. It is frequently deleted and inactivated in prostate cancer and may exert a tumor suppressor function. However, how KLF5 inhibits the progression of prostate cancer is still not clear. In the present study, we identified how KLF5 and tumor necrosis factor α (TNFα) pathway, which can induce apoptosis in cancer, regulate each other in LNCaP prostate cancer cells.Material and Methods: The expression of messenger RNA and protein was detected by real-time polymerase chain reaction assay and western blot analysis, respectively. To identify whether KLF5 regulates the activity of TNFα downstream pathway, we constructed a stable KLF5 knockdown or KLF5 overexpressing cell line with lentivirus-containing short hairpin RNA targeting KLF5 or full-length KLF5 in LNCaP cells. Cell apoptosis was determined through flow cytometry assay. In addition, the regulation of KLF5 on target gene transcription was detected by reporter luciferase activity assay, and the binding of KLF5 on target promoter was detected through oligonucleotides pull-down analysis.Results: We found that TNFα induced the expression of KLF5 at both messenger RNA and protein levels; moreover, TNFα up-regulated KLF5 through TNF receptor 1 but not through TNF receptor 2 in LNCaP cells. Knockdown of KLF5 decreased apoptosis induced by TNFα, whereas cell apoptosis was increased by KLF5 overexpression. Consistently, expression of cleaved PARP and caspase-3 induced by TNFα was decreased by KLF5 knockdown, whereas it was increased by overexpressed KLF5. JNK activity is essential for the apoptosis induced by TNFα. We found that knockdown of KLF5 not only decreased the phosphorylation of JNK induced by TNFα, but also down-regulated the transcription of mitogen-activated protein kinase kinase 7 (MKK7), an upstream kinase of JNK, by binding to the MKK7 promoter.Conclusions: Our results indicate that KLF5 is an essential transcription regulator of MKK7 kinase and promotes the apoptosis induced by TNFα in LNCaP cells. Loss of KLF5 in prostate cancer may decrease cell response to TNFα-inducing apoptosis and facilitate cancer initiation and progression; moreover, KLF5 could be a potential molecular marker for predicting the effect of high-dose TNFα on tumor growth inhibition in prostate cancer. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase kinase 7 (MKK7) is a key signal transduction regulator in c-Jun N-terminal kinase (JNK) signaling pathway, which is involved in a wide range of physiological and pathological processes. In this study, we described the molecular cloning of a new member of MKK7 group from Litopenaeus vannamei named as LvMKK7. The full-length cDNA of LvMKK7 was 3093 bp in length, with an open reading frame (ORF) of 1440 bp encoding a putative protein of 479 amino acids. LvMKK7 contained a conserved kinase domain of 261 amino acids in which there was a characteristic S-K-A-K-T motif as a potential target site of phosphorylation by MKKK. Moreover, subcellular localization showed LvMKK7 was located in both the cytoplasm and the nucleus of Drosophila S2 cells. Real-time PCR indicated that LvMKK7 was universally expressed in all tested tissues and its expression in hepatopancreas was responsive to the challenge of LPS, Poly (I:C), Vibrio parahaemolyticus , Staphhylococcus aureus and white spot syndrome virus (WSSV). In addition, co-immunoprecipitation assay demonstrated that LvJNK was phosphorylated and activated by LvMKK7, which suggested LvMKK7 was the upper regulator of LvJNK. Furthermore, RNAi-mediated knockdown of LvMKK7 enhanced the sensitivity of shrimps to V. parahaemolyticus infection. Overall, our results suggested that LvMKK7 may play important roles in the shrimp innate immunity. [ABSTRACT FROM AUTHOR]

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ~25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling. [ABSTRACT FROM AUTHOR]

In this study, we investigated the molecular mechanisms underlying the anti-inflammatory effects of α-chaconine in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in LPS-induced septic mice. α-Chaconine inhibited the expressions of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at the transcriptional level, and attenuated the transcriptional activity of activator protein-1 (AP-1) by reducing the translocation and phosphorylation of c-Jun. α-Chaconine also suppressed the phosphorylation of TGF-β-activated kinase-1 (TAK1), which lies upstream of mitogen-activated protein kinase kinase 7 (MKK7)/Jun N-terminal kinase (JNK) signaling. JNK knockdown using siRNA prevented the α-chaconine-mediated inhibition of pro-inflammatory mediators. In a sepsis model, pretreatment with α-chaconine reduced the LPS-induced lethality and the mRNA and production levels of pro-inflammatory mediators by inhibiting c-Jun activation. These results suggest that the anti-inflammatory effects of α-chaconine are associated with the suppression of AP-1, and support its possible therapeutic role for the treatment of sepsis. [ABSTRACT FROM AUTHOR]

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSSinduced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N"-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdownof apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and autophosphorylation of c-Src. The present study demonstrates that Ca2+ /Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSSinduced tight junction disruption and barrier dysfunction. [ABSTRACT FROM AUTHOR]

Metastasis is the process of primary tumor cells breaking away and colonizing distant secondary sites. In order for a tumor cell growing in one microenvironment to travel to, and flourish in, a secondary environment, it must survive a series of events termed the metastatic cascade. Before departing the primary tumor, cells acquire genetic and epigenetic changes that endow them with properties not usually associated with related normal differentiated cells. Those cells also induce a subset of bone marrow-derived stem cells to mobilize and establish pre-metastatic niches []. Many tumor cells undergo epithelial-to-mesenchymal transition (EMT), where they transiently acquire morphologic changes, reduced requirements for cell-cell contact and become more invasive []. Invasive tumor cells eventually enter the circulatory (hematogenous) or lymphatic systems or travel across body cavities. In transit, tumor cells must resist anoikis, survive sheer forces and evade detection by the immune system. For blood-borne metastases, surviving cells then arrest or adhere to endothelial linings before either proliferating or extravasating. Eventually, tumor cells complete the process by proliferating to form a macroscopic mass []. Up to 90 % of all cancer related morbidity and mortality can be attributed to metastasis. Surgery manages to ablate most primary tumors, especially when combined with chemotherapy and radiation. But if cells have disseminated, survival rates drop precipitously. While multiple parameters of the primary tumor are predictive of local or distant relapse, biopsies remain an imperfect science. The introduction of molecular and other biomarkers [, ] continue to improve the accuracy of prognosis. However, the invasive procedure introduces new complications for the patient. Likewise, the heterogeneity of any tumor population [, , ] means that sampling error (i.e., since it is impractical to examine the entire tumor) necessitates further improvements. In the case of breast cancer, for example, women diagnosed with stage I diseases (i.e., no evidence of invasion through a basement membrane) still have a ~30 % likelihood of developing distant metastases []. Many physicians and patients opt for additional chemotherapy in order to 'mop up' cells that have disseminated and have the potential to grow into macroscopic metastases. This means that ~ 70 % of patients receive unnecessary therapy, which has undesirable side effects. Therefore, improving prognostic capability is highly desirable. Recent advances allow profiling of primary tumor DNA sequences and gene expression patterns to define a so-called metastatic signature [-], which can be predictive of patient outcome. However, the genetic changes that a tumor cell must undergo to survive the initial events of the metastatic cascade and colonize a second location belie a plasticity that may not be adequately captured in a sampling of heterogeneous tumors. In order to tailor or personalize patient treatments, a more accurate assessment of the genetic profile in the metastases is needed. Biopsy of each individual metastasis is not practical, safe, nor particularly cost-effective. In recent years, there has been a resurrection of the notion to do a 'liquid biopsy,' which essentially involves sampling of circulating tumor cells (CTC) and/or cell free nucleic acids (cfDNA, including microRNA (miRNA)) present in blood and lymph [-]. The rationale for liquid biopsy is that tumors shed cells and/or genetic fragments into the circulation, theoretically making the blood representative of not only the primary tumor but also distant metastases. Logically, one would predict that the proportion of CTC and/or cfDNA would be proportionate to the likelihood of developing metastases []. While a linear relationship does not exist, the information within CTC or cfDNA is beginning to show great promise for enabling a global snapshot of the disease. However, the CTC and cfDNA are present at extremely low levels. Nonetheless, newer technologies capture enough material to enrich and sequence the patient's DNA or quantification of some biomarkers. Among the biomarkers showing great promise are metastasis suppressors which, by definition, block a tumor cell's ability to complete the metastatic process without prohibiting primary tumor growth []. Since the discovery of the first metastasis suppressor, Nm23, more than 30 have been functionally characterized. They function at various stages of the metastatic cascade, but their mechanisms of action, for the most part, remain ill-defined. Deciphering the molecular interactions of functional metastasis suppressors may provide insights for targeted therapies when these regulators cease to function and result in metastatic disease. In this brief review, we summarize what is known about the various metastasis suppressors and their functions at individual steps of the metastatic cascade (Table 1). Some of the subdivisions are rather arbitrary in nature, since many metastasis suppressors affect more than one step in the metastatic cascade. Nonetheless what emerges is a realization that metastasis suppressors are intimately associated with the microenvironments in which cancer cells find themselves []. [ABSTRACT FROM AUTHOR]

The prognosis of advanced colon cancer patients is profoundly affected by the presence or absence of liver metastasis. miR-493 functions as a potent suppressor of liver metastasis, and low-level miR-493 expression in human primary colon cancer is associated with an elevated incidence of liver metastasis. We previously showed that IGF1R is a target gene of miR-493, and that the inhibition of IGF1R partly explains how miR-493 suppresses liver metastasis. However, major functional targets that mediate the antimetastatic activity of miR-493 remain elusive. Here, we extended our search for target genes and identified MKK7, a mitogen-activated protein kinase kinase, as a novel target of miR-493. miR-493 inhibits MKK7 expression by targeting the binding site at the 3′- UTR of the mkk7 gene. MKK7 was expressed in six out of seven colon cancer cell lines examined but not in non-transformed colon epithelial cells, and its expression was required for the activating phosphorylation of JNK. RNA interference-mediated inhibition of MKK7 resulted in marked suppression of liver metastasis of colon cancer cells. A significant decrease of metastasized cells by the MKK7 knockdown was observed, even at early stages of the metastatic settlement, in accordance with a time course of the miR-493-mediated inhibition of the metastasis. Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7. Thus, MKK7 is a major functional target of miR-493, and its suppression thwarts liver metastasis of colon cancer cells. [ABSTRACT FROM AUTHOR]

In vivo gene knockout studies in mice have revealed essential roles of the mitogen-activated protein kinases (MAPKs) in embryogenesis, but due to early lethality of the knockout embryos, the underlying mechanisms and specific developmental programs regulated by the MAPK pathways have remained largely unknown. In vitro differentiation of mouse embryonic stem cells (ESCs) have opened new possibilities for understanding lineage segregation and gene function in the developmental stages that are not normally accessible in vivo. Building on this technology, in combination with gene knockout cells, we investigated the roles of MKK4 and MKK7, two upstream kinases of the MAPKs, in early lineage specification. Our results show that MKK4 and MKK7 differentially regulate the JNK and p38 MAPKs and make distinct contributions to differentiation programs. In vitro ESC differentiation is a valuable system to investigate the molecular and signaling mechanisms of early embryogenesis. [ABSTRACT FROM AUTHOR]

Chronic aristolochic acid nephropathy (CAAN) is a chronic and progressive tubulointerstitial nephropathy characterized by extensive interstitial fibrosis. Aristolochic acid (AA) could induce overexpression of transforming growth factor-β1 (TGF-β1) in a human renal proximal tubule epithelial cells line (HKC), which has been implicated in the pathogenesis of CAAN. The present studies in HKC cells showed 1) AA could activate JNK in time- and dose-dependent manners and JNK inhibitor SP600125 could inhibit AA-induced TGF-β1 promoter activity and TGF-β1 synthesis; 2) AA-induced JNK activation and TGF-β1 synthesis were significantly inhibited by kinase-inactive mutants of MEKK4, MKK4, or MKK7; 3) AA could upregulate luciferase activity derived by a wild-type TGF-β1 promoter, but not by an AP-1 binding-deficient TGF-β1 promoter; and 4) AA could upregulate expression of c-Fos, phospho-c-Jun, and phospho-ATF2. The above data suggest AA-induced TGF-β1 overexpression in HKC cells may be mainly mediated by the JNK signaling pathway. Both the upstream kinases of JNK including MEKK4, MKK4, and MKK7, and the downstream transcription factor of JNK, AP-1, may also participate in this process. [ABSTRACT FROM AUTHOR]

Abstract: c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1, which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes. [Copyright &y& Elsevier]

The Liver X Receptor (LXR) and Pregnane X Receptor (PXR) are members of the nuclear receptor superfamily. Previously, they have been classified as important regulators of lipid homeostasis. However, recent studies have shown that they may be implicated in anti-inflammatory responses as well. This study shows that Tumour Necrosis Factor-a (TNF-α) treatment reduces both LXR-α and PXR mRNA expression. However, pre-treatment with rapamycin, an mTOR inhibitor, followed by TNF-α stimulation, significantly induces LXR-α and PXR mRNA expression to ~17- and ~2- fold, respectively. This suggests that mTORC1, a multi-molecular complex of which mTOR is a member, may act as a negative regulator that inhibits the induction of LXR-α and PXRas anti- inflammatory genes. It is also shown here that inhibition of JNK1 via the mTOR/Akt pathway coincides with the up-regulation of LXR-α and PXR mRNA, after TNF-α treatment. Together, these observations suggest that JNK1 possibly act downstream of mTORC1 as an LXR-α and PXR inhibitor. From the results gleaned in this study, rapamycin (and its analogues) may be used to reduce acute inflammation by promoting the induction of LXR-a and PXR as anti-inflammatory genes. [ABSTRACT FROM AUTHOR]

Abstract: The functions of mitogen-activated protein kinases (MKKs) 4 and 7 are typically associated with the c-Jun N-terminal kinase (JNK) signaling pathway. Both MKKs synergistically phosphorylate different JNK isoforms and are therefore involved in numerous physiological (e.g. differentiation and proliferation) and pathological (e.g. apoptosis and tumorigenesis) processes. MKK4 and MKK7 share similar molecular characteristics as well as several upstream activators and scaffold proteins. However, their functions are non-redundant and determined by different stimuli, biochemical interactions and differential tissue distribution. The central question is how two MKKs regulate or affect the multiple actions of their JNK substrates. Similar to JNKs, MKK4 and MKK7 can simultaneously exert divergent functions in different cellular compartments and signalosomes. It is also important to realize that the MKK effects are splice variant-specific. The present review not only summarizes the various modes of MKK4 and MKK7 activation and activity, but also their functions. We also extensively describe their impact on JNK signaling, their molecular interactions resulting in the formation of context-specific signalosomes and the functional consequences of JNK deficiency. [Copyright &y& Elsevier]

Abstract: The c-Jun N-terminal kinases (JNKs) mediate a diversity of physiological and pathophysiological effects. Apart from isoform-specific JNK activation, upstream kinases are supposed to be the relevant regulators, which are involved in the context- and signalosome-depending functions. In the present study we report the cloning and characterization of the novel rat MKK7γ1, a splice variant of MKK7 with an additional exon in the N-terminal region, in the neuronal pheochromocytoma cell line PC12. Transfected MKK7γ1 increased basal JNK activity, in particular phosphorylation of JNK2. Consequently, JNK signalling was changed in mRNA-, protein- and activation-levels of JNK targets, such as transcription factors (c-Jun, p53, c-Myc), cell cycle regulators (p21, CyclinD1) and apoptotic proteins (Fas, Bim, Bcl-2, Bcl-xl). These alterations promote the sensitivity of MKK7γ1-transfected cells towards cell death and repress cell proliferation under normal cell growth conditions. Complexes of JIP-1, MKK7 and JNK2 were the major JNK signalosomes under basal conditions. After stimulation with taxol (5μM) and tunicamycin (1.4μg/ml), MKK7γ1- but not MKK7β1-transfection, reduced cell death and even increased cell proliferation. Cellular stress also led to an increased phosphorylation of JNK1 and the almost complete abrogation of complexes of JIP-1, MKK7 and JNK2 in MKK7γ1-transfected PC12 cells. Summarizing, MKK7γ1 affects the function and activity of individual JNK isoforms and the formation of their signalosomes. This study demonstrates for the first time that one splice-variant of MKK7 tightly controls JNK signalling and effectively adapts JNK functions to the cellular context. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase kinases (MAPKKs) are important components of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signalling pathway. Two MAPKKs that are crucial transducers upstream of JNK signalling are MKK4 and MKK7. These two MAPKKs directly phosphorylate specific Tyr and Thr residues located in the activation loop of the JNK protein and activate this kinase in response to environmental stress, pro-inflammatory cytokines or developmental cues. Although much is known about the biochemical and structural bases of the catalytic mechanism of the MAPKKs, the regulation and physiological functions of these enzymes during early embryogenesis have remained a mystery until relatively recently. Studies employing a range of animal models have now revealed the essential roles that MAPKKs play in diverse developmental contexts, including in dorsoventral patterning, convergent extension and somitogenesis. Focusing primarily on extensive work done in mouse and zebrafish models, this review summarizes the functional properties of MKK4 and MKK7 during vertebrate and invertebrate development, and the mechanisms by which these kinases regulate multiple steps in the establishment of the body plan of an organism. [ABSTRACT FROM PUBLISHER]

Autosomal dominant mutations in the human Leucine-Rich Repeat Kinase 2 ( LRRK2) gene represent the most common monogenetic cause of Parkinson disease (PD) and increased kinase activity observed in pathogenic mutants of LRRK2 is most likely causative for PD-associated neurotoxicity. The sequence of the LRRK2 kinase domain shows similarity to MAP kinase kinase kinases. Furthermore, LRRK2 shares highest sequence homology with mixed linage kinases which act upstream of canonical MAPKK and are involved in cellular stress responses. Therefore, we addressed the question if LRRK2 exhibits MAPKKK activity by systematically testing MAPKKs as candidate substrates, in vitro. We demonstrate that LRRK2 variants phosphorylate mitogen-activated protein kinase kinases (MAPKK), including MKK3 -4, -6 and -7. MKKs act upstream of the MAPK p38 and JNK mediating oxidative cell stress, neurotoxicity and apoptosis. The disease-associated LRRK2 G2019S and I2020T mutations show an increased phosphotransferase activity towards MKKs correlating with the activity shown for its autophosphorylation. Our findings present evidence of a new class of molecular targets for mutant LRRK2 that link to neurotoxicity, cellular stress, cytoskeletal dynamics and vesicular transport. [ABSTRACT FROM AUTHOR]