Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Cyano Enone-Bearing Triterpenoid Soloxolone Methyl Inhibits Epithelial-Mesenchymal Transition of Human Lung Adenocarcinoma Cells In Vitro and Metastasis of Murine Melanoma In Vivo.

Introduction of α-cyano α,ß-unsaturated carbonyl moiety into natural cyclic compounds markedly improves their bioactivities, including inhibitory potential against tumor growth and metastasis. Previously, we showed that cyano enone-bearing derivatives of 18ßH-glycyrrhetinic (GA) and deoxycholic acids displayed marked cytotoxicity in different tumor cell lines. Moreover, GA derivative soloxolone methyl (SM) was found to induce ER stress and apoptosis in tumor cells in vitro and inhibit growth of carcinoma Krebs-2 in vivo. In this work, we studied the effects of these compounds used in non-toxic dosage on the processes associated with metastatic potential of tumor cells. Performed screening revealed SM as a hit compound, which inhibits motility of murine melanoma B16 and human lung adenocarcinoma A549 cells and significantly suppresses colony formation of A549 cells. Further study showed that SM effectively blocked transforming growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 cells: namely, inhibited TGF-ß-stimulated motility and invasion of tumor cells as well as loss of their epithelial characteristics, such as, an acquisition of spindle-like phenotype, up- and down-regulation of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin, zona occludens-1 (ZO-1)) markers, respectively. Network pharmacology analysis with subsequent verification by molecular modeling revealed that matrix metalloproteinases MMP-2/-9 and c-Jun N-terminal protein kinase 1 (JNK1) can be considered as hypothetical primary targets of SM, mediating its marked anti-EMT activity. The inhibitory effect of SM on EMT revealed in vitro was further confirmed in a metastatic model of murine B16 melanoma: SM was found to effectively block metastatic dissemination of melanoma B16 cells in vivo, increase expression of E-cadherin and suppress expression of MMP-9 in lung metastatic foci. Altogether, our data provided valuable information for a better understanding of the antitumor activity of cyano enone-bearing semisynthetic compounds and revealed SM as a promising anti-metastatic drug candidate.

EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression.

The members of human miR-17-92 cluster are implicated in several cancers and are known to increase cancer cells invasiveness. The present study reports reduced expression of miR-92a-1-5p in EGF treated HTR-8/SVneo trophoblastic cells by NGS and qRT-PCR. Overexpression of miR-92a-1-5p led to significantly reduced EGF-mediated HTR-8/SVneo cells invasion. MAPK8 and FAS were predicted to be miR-92a-1-5p targets, and confirmed to be reduced by qRT-PCR and Western blotting in trophoblast cells overexpressing miR-92a-1-5p. The binding of miR-92a-1-5p to MAPK8 and FAS 3'-UTR was confirmed by Luciferase reporter assay and Rescue assay. EGF increases MMP-2 & MMP-9 expression and reduces TIMP1 expression in HTR-8/SVneo cells. Inhibition of MAPK8 (by SP600125) reduced EGF-mediated MMP-9/TIMP1 ratio and invasion. Similarly, silencing of FAS by siRNA reduced EGF-mediated MMP-2/TIMP1 ratio and invasion. Treatment of HTR-8/SVneo cells with STAT1/3 inhibitors or siRNAs led to loss of EGF-mediated reduction in miR-92a-1-5p levels. Inserting the predicted binding sites of STAT3 present in promoter region of miR-92a-1-5p upstream of Luciferase promoter reduced its expression in presence of STAT3 expression vector. Thus, EGF leads to reduced miR-92a-1-5p expression which may be regulated by STAT1/STAT3 and controls HTR-8/SVneo cells invasion by targeting MAPK8 and FAS, which in turn increases MMP-2/MMP-9 expression.

Irreversible JNK1-JUN inhibition by JNK-IN-8 sensitizes pancreatic cancer to 5-FU/FOLFOX chemotherapy.

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.

Integrative discovery of treatments for high-risk neuroblastoma.

Despite advances in the molecular exploration of paediatric cancers, approximately 50% of children with high-risk neuroblastoma lack effective treatment. To identify therapeutic options for this group of high-risk patients, we combine predictive data mining with experimental evaluation in patient-derived xenograft cells. Our proposed algorithm, TargetTranslator, integrates data from tumour biobanks, pharmacological databases, and cellular networks to predict how targeted interventions affect mRNA signatures associated with high patient risk or disease processes. We find more than 80 targets to be associated with neuroblastoma risk and differentiation signatures. Selected targets are evaluated in cell lines derived from high-risk patients to demonstrate reversal of risk signatures and malignant phenotypes. Using neuroblastoma xenograft models, we establish CNR2 and MAPK8 as promising candidates for the treatment of high-risk neuroblastoma. We expect that our method, available as a public tool (targettranslator.org), will enhance and expedite the discovery of risk-associated targets for paediatric and adult cancers.

CLEFMA Activates the Extrinsic and Intrinsic Apoptotic Processes through JNK1/2 and p38 Pathways in Human Osteosarcoma Cells.

Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA's increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA's apoptotic effects on human osteosarcoma cells.

The peroxidase PRDX1 inhibits the activated phenotype in mammary fibroblasts through regulating c-Jun N-terminal kinases.

BACKGROUND: Reactive oxygen species (ROS), including hydrogen peroxide, drive differentiation of normal fibroblasts into activated fibroblasts, which can generate high amounts of hydrogen peroxide themselves, thereby increasing oxidative stress in the microenvironment. This way, activated fibroblasts can transition into cancer-associated fibroblasts (CAFs). METHODS: Mammary fibroblasts from either female 8 weeks old PRDX1 knockout and wildtype mice or Balb/c mice were studied for characteristic protein expression using immunofluorescence and immunoblotting. Cancer-associated fibroblasts was examined by transwell migration and invasion assays. The binding of PRDX1 to JNK1 was assessed by co-immuneprecipitation and JNK regulation of CAF phenotypes was examined using the JNK inhibitor SP600125. Extracellular hydrogen peroxide levels were measured by chemiluminescence via the reaction between hypochlorite and luminol. Statistical analyses were done using Students t-test. RESULTS: We show here PRDX1 activity as an essential switch in regulating the activated phenotype as loss of PRDX1 results in the development of a CAF-like phenotype in mammary fibroblasts. We also show that PRDX1 regulates JNK kinase signaling thereby inhibiting CAF-like markers and CAF invasion. Inhibition of JNK activity reduced these behaviors. CONCLUSIONS: These data suggest that PRDX1 repressed the activated phenotype of fibroblasts in part through JNK inhibition which may present a novel therapeutic option for CAF-enriched cancers such as breast cancer.

JNK1 regulates RANKL-induced osteoclastogenesis via activation of a novel Bcl-2-Beclin1-autophagy pathway.

JNK1 plays an important role in osteoclastogenesis in response to the osteoclastogenic cytokine receptor activator for nuclear factor-κB ligand (RANKL). JNK1 is widely accepted as an autophagy regulator under stress conditions. However, the role of JNK1-mediated autophagy in osteoclastogenesis remains largely unknown. In the current study, our data showed that JNK1 inhibition by a pharmacological inhibitor or RNA interference significantly reduced the autophagic response induced by RANKL in osteoclast precursors (OCPs) derived from bone marrow-derived macrophages. Overexpression of the key autophagy protein Beclin1 rescued autophagy deficiency and osteoclastogenesis in the presence of a JNK inhibitor (SP600125). In contrast, JNK activator (anisomycin)-induced autophagy was blocked by Beclin1 knockdown in OCPs. In addition, JNK1 inhibition increased apoptosis and blocked autophagy, whereas overexpression of Beclin1 reversed the enhanced apoptosis induced by JNK1 inhibition in OCPs. Furthermore, RANKL could induce the phosphorylation of Bcl-2, subsequently dissociating Beclin1 from the Bcl-2-Beclin1 complex, which could be blocked by JNK1 inhibition. Collectively, this study revealed that JNK1 regulated osteoclastogenesis by activating Bcl-2-Beclin1-autophagy signaling in addition to the classic c-Jun/activator protein 1 pathway, which provided the first evidence for the contribution of JNK1 signaling to OCP autophagy and the autophagic mechanism underlying JNK1-regulated osteoclastogenesis. An important osteoclastogenesis-regulating signaling pathway (JNK1-Bcl-2-Beclin1-autophagy activation) was identified, which provides novel potential targets for the clinical therapy of metabolic bone diseases.-Ke, D., Ji, L., Wang, Y., Fu, X., Chen, J., Wang, F., Zhao, D., Xue, Y., Lan, X., Hou, J. JNK1 regulates RANKL-induced osteoclastogenesis via activation of a novel Bcl-2-Beclin1-autophagy pathway.

c­Jun N­terminal kinase inhibition attenuates early brain injury induced neuronal apoptosis via decreasing p53 phosphorylation and mitochondrial apoptotic pathway activation in subarachnoid hemorrhage rats.

Early brain injury (EBI)­induced neuronal apoptosis is primarily responsible for the subsequent complications of aneurysmal subarachnoid hemorrhage (aSAH), which may increase the risk of mortality in patients with aSAH. c­Jun N­terminal kinase (JNK) has been demonstrated to be a promoter of EBI­induced cell apoptosis, although the mechanism has yet to be fully elucidated. The present study aimed to explore whether the role of JNK1 is associated with tumor protein p53 (p53), which is one of the most important factor that triggers cell apoptosis. JNK1 expression was downregulated via in vivo small interfering RNA transfection in an aSAH rat model in order to assess differences in the behavior, survival times, morphology and genetics of the experimental animals. The results revealed that JNK1 inhibition improved the neurological scores and survival times of SAH rats by interrupting cascaded neuronal apoptosis. The interruption of EBI­induced neuronal apoptosis may originate from a decrease in the level of p53 phosphorylation and deactivation of the downstream mitochondrial apoptotic pathway. Taken together, these results suggest that JNK1 may be a promising target for improving the prognosis of patients with aSAH.

Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-ß signaling results in variable activation of human dermal fibroblasts.

Fibroblast activation is a key step in the establishment of skin fibrosis induced by acute injury, and it is characterized by the differentiation of plastic resident tissue fibroblasts into contractile, extracellular matrix­secreting myofibroblasts. As fibroblast activation must be regulated in vivo, fibroblasts receive signals from the surrounding environment that initiate their fibrotic program. Thus, the present study investigated the effects of mitogen­activated protein kinase (MAPK) signaling pathways on fibroblast activation. It was demonstrated in primary human dermal fibroblasts that small molecule­mediated inhibition of extracellular signal­regulated kinase (ERK) and c­Jun N­terminal kinase (JNK) potentiated fibroblast activation, and that small molecule­mediated inhibition of p38 antagonized fibroblast activation. ERK and JNK inhibition cooperatively enhanced fibroblast activation mediated by treatment with exogenous transforming growth factor (TGF)­ß1, and p38 inhibition antagonized ERK inhibitor­mediated or JNK inhibitor­mediated fibroblast activation. Transcript analysis demonstrated that ERK and JNK inhibitor­mediated fibroblast activation was accompanied by distinct changes in the expression of TGF­ß­associated ligands and receptors, and that p38 inhibitor­mediated antagonism of fibroblast activation was accompanied by a distinct expression paradigm of TGF­ß­associated genes, including upregulation of betaglycan. ERK inhibitor­mediated and JNK inhibitor­mediated fibroblast activation was partially antagonized by small molecule­mediated inhibition of TGF­ß receptor (R)1, indicating that these mechanisms of fibroblast activation are partially dependent on TGF­ß/TGF­ßR signaling. These data collectively demonstrate and provide partial explanations of the varied effects and pathway dependencies of MAPK inhibitor­mediated effects on fibroblast activation.

Curcumol induces RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling in hepatic stellate cells.

It is generally recognized that hepatic fibrogenesis is an end result of increased extracellular matrix (ECM) production from the activation and proliferation of hepatic stellate cells (HSCs). An in-depth understanding of the mechanisms of HSC necroptosis might provide a new therapeutic strategy for prevention and treatment of hepatic fibrosis. In this study, we attempted to investigate the effect of curcumol on necroptosis in HSCs, and further to explore the molecular mechanisms. We found that curcumol ameliorated the carbon tetrachloride (CCl4)-induced mice liver fibrosis and suppressed HSC proliferation and activation, which was associated with regulating HSC necroptosis through increasing the phosphorylation of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3). Moreover, curcumol promoted the migration of RIPK1 and RIPK3 into necrosome in HSCs. RIPK3 depletion impaired the anti-fibrotic effect of curcumol. Importantly, we showed that curcumol-induced RIPK3 up-regulation significantly increased mitochondrial reactive oxygen species (ROS) production and mitochondrial depolarization. ROS scavenger, N-acetyl-L-cysteine (NAC) impaired RIPK3-mediated necroptosis. In addition, our study also identified that the activation of c-Jun N-terminal kinase1/2 (JNK1/2) was regulated by RIPK3, which mediated curcumol-induced ROS production. Down-regulation of RIPK3 expression, using siRIPK3, markedly abrogated JNK1/2 expression. The use of specific JNK1/2 inhibitor (SP600125) resulted in the suppression of curcumol-induced ROS production and mitochondrial depolarization, which in turn, contributed to the inhibition of curcumol-triggered necroptosis. In summary, our study results reveal the molecular mechanism of curcumol-induced HSC necroptosis, and suggest a potential clinical use of curcumol-targeted RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling for the treatment of hepatic fibrosis.

Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1.

Colorectal cancer is one of the most commonly diagnosed cancers and the third most common cause of cancer-related death. Metastasis is the leading reason for the resultant mortality of these patients. Accordingly, development and characterization of novel anti-cancer drugs limiting colorectal tumor cell dissemination and metastasis are needed. In this study, we found that the small molecule Reversine reduces the migration potential of human colon carcinoma cells in vitro. A coupled kinase assay with bio-informatics approach identified the c-Jun N-terminal kinase (JNK) cascade as the main pathway inhibited by Reversine. Knockdown experiments and pharmacological inhibition identified JNK1 but not JNK2, as a downstream effector target in cancer cell migration. Xenograft experiments confirm the effect of JNK inhibition in the metastatic potential of colon cancer cells. These results highlight the impact of individual JNK isoforms in cancer cell metastasis and propose Reversine as a novel anti-cancer molecule for treatment of colon cancer patients.

Involvement of JNK1/2-NF-κBp65 in the regulation of HMGB2 in myocardial ischemia/reperfusion-induced apoptosis in human AC16 cardiomyocytes.

JNK1/2 and NF-κB signal are essential signaling pathways that mediate a variety of cellular processes, including cell survival, apoptosis, inflammation and angiogenesis. JNK1/2 activation and NF-κBp65 nuclear translocation have been found in ischemia/reperfusion (I/R)-induced injury. However, the regulation of JNK1/2-NF-κBp65 signaling pathway remains unclear. To examine the function and possible mechanism of HMGB2 in I/R-induced cell injury, human AC16 cardiomyocytes transfected with pLVX-Puro-HMGB2 were treated with SP600125 (JNK1/2 inhibitor) or PDTC (NF-κB inhibitor) and that following I/R injury were transfected with HMGB2-shRNA. The cell proliferation and apoptosis were measured by CCK-8, flow cytometry and TUNEL, respectively. The expression of HMGB2, Cleaved PARP and Caspase-3, Bax and Bcl-2 and activity of MAPKs and NF-κBp65 were measured by Western blot. Here, we found that I/R time-dependently induced the increase in the expression of HMGB2 in AC16 cardiomyocytes. HMGB2 silencing significantly inhibited I/R-induced the cell proliferation reduction, cell apoptosis, activation of ERK1/2, JNK1/2 and NF-κBp65, increased Bax, Cleaved PARP and Caspase-3 and decreased Bcl-2 expression. HMBG2 overexpression mimicked the effect of I/R-induced injury in AC16 cardiomyocytes, which was reversed by treatment with SP600125 or PDTC. Moreover, PDTC treatment in rats following I/R injury also showed decreases in the cell apoptosis, HMGB2, Cleaved PARP and Caspase-3 and Bax expression, and JNK1/2 activation. Taken together, our findings suggest that HMBG2 overexpression promotes I/R-induced cell apoptosis through activating the JNK1/2-NF-κBp65 signaling in AC16 cardiomyocytes.

Role of Fibroblast Growth Factor Receptor 2b in the Cross Talk between Autophagy and Differentiation: Involvement of Jun N-Terminal Protein Kinase Signaling.

Fibroblast growth factor receptor 2b (FGFR2b) is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of FGFR2b-mediated early differentiation of keratinocytes. However, the molecular mechanisms regulating this interplay remain to be elucidated. Since we have also recently shown that Jun N-terminal protein kinase 1 (JNK1) signaling is involved in FGFR2b-induced autophagy and a possible role of the JNK pathway in epidermal differentiation has been suggested (though it is still debated), we investigated here the cross talk between FGFR2b-mediated autophagy and differentiation, focusing on the downstream JNK signaling. Biochemical, molecular, and immunofluorescence approaches in 2-dimensional (2-D) keratinocyte cultures and three-dimensional (3-D) organotypic skin equivalents confirmed that FGFR2b overexpression increased both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering of early differentiation. In contrast, the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway did not appear to be involved in the two processes, and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy and differentiation.

3'-Hydroxy-3,4'-dimethoxyflavone-induced cell death in human leukaemia cells is dependent on caspases and reactive oxygen species and attenuated by the inhibition of JNK/SAPK.

Flavonoids are phenolic substances that appear to exert beneficial effects in several chronic diseases, including cancer. Structure-activity relationships of the cytotoxic activity of a series of flavonols and their 3-methyl ether derivatives established that 3'-hydroxy-3,4'-dimethoxyflavone (flavonoid 11) displayed strong cytotoxicity against human leukaemia cell lines (HL-60, U-937 and MOLT-3), and cells that over-express the anti-apoptotic proteins, Bcl-2 and Bcl-xL, and against P-glycoprotein-overexpressing K-562/ADR cells. This compound induced G2-M cell cycle arrest and it was a potent apoptotic inducer on HL-60, MOLT-3, U-937 and U-937/Bcl-2 cell lines. Cell death was (i) mediated by caspase activation, since it was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by a selective caspase-9 inhibitor, (ii) associated with cytochrome c release, the dissipation of the inner mitochondrial membrane potential (ΔΨm) and the activation of the mitogen-activated protein kinase pathway and (iii) partially blocked by the inhibition of c-jun NH2 terminal kinases/stress activated protein kinases (JNK/SAPK) signalling and by the free-radical scavenger N-acetyl-l-cysteine.

JNK1 inhibition by Licochalcone A leads to neuronal protection against excitotoxic insults derived of kainic acid.

The mitogen-activated protein kinase family (MAPK) is an important group of enzymes involved in cellular responses to diverse external stimuli. One of the members of this family is the c-Jun-N-terminal kinase (JNK). The activation of the JNK pathway has been largely associated with the pathogenesis that occurs in epilepsy and neurodegeneration. Kainic acid (KA) administration in rodents is an experimental approach that induces status epilepticus (SE) and replicates many of the phenomenological features of human temporal lobe epilepsy (TLE). Recent studies in our group have evidenced that the absence of the JNK1 gene has neuroprotective effects against the damage induced by KA, as it occurs with the absence of JNK3. The aim of the present study was to analyse whether the pharmacological inhibition of JNK1 by Licochalcone A (Lic-A) had similar effects and if it may be considered as a new molecule for the treatment of SE. In order to achieve this objective, animals were pre-treated with Lic-A and posteriorly administered with KA as a model for TLE. In addition, a comparative study with KA was performed between wild type pre-treated with Lic-A and single knock-out transgenic mice for the Jnk1-/- gene. Our results showed that JNK1 inhibition by Lic-A, previous to KA administration, caused a reduction in the convulsive pattern. Furthermore, it reduced phosphorylation levels of the JNK, as well as its activity. In addition, Lic-A prevented hippocampal neuronal degeneration, increased pro-survival anti-apoptotic mechanisms, reduced pro-apoptotic biomarkers, decreased cellular stress and neuroinflammatory processes. Thus, our results suggest that inhibition of the JNK1 by Lic-A has neuroprotective effects and that; it could be a new potential approach for the treatment of SE and neurodegeneration.

Structural Basis for the Selective Inhibition of c-Jun N-Terminal Kinase 1 Determined by Rigid DARPin-DARPin Fusions.

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.

The p38 Mitogen-Activated Protein Kinase Critically Regulates Human Keratinocyte Inflammasome Activation.

Inflammasomes are key intracellular signaling platforms involved in innate immune responses to micro-organisms and danger signals. Extracellular signal-regulated kinase, Jun N-terminal kinase, and p38 mitogen-activated protein kinase family members are activated by numerous environmental stresses. Recently, it has been reported that Jun N-terminal kinase is involved in inflammasome activation in myeloid immune cells. To date, the role of mitogen-activated protein kinase in inflammasome activity in keratinocytes has not been investigated. Here, we show that, in primary human keratinocytes, p38 mitogen-activated protein kinase is required for inflammasome activation and IL-1ß secretion. Using selective small molecule inhibitors, small interfering RNA gene silencing, and CRISPR/Cas9-based deletion, we demonstrate the above and identify p38α and p38δ as critical regulators of ASC oligomerization, inflammasome activation, and IL-1ß secretion in keratinocytes. Furthermore, our data suggest that the nature of the mitogen-activated protein kinase regulating inflammasome activity exhibits a certain cell specificity, with p38 playing a predominant role in keratinocytes and Jun N-terminal kinase 1 in cells of myeloid origin.

GLP-1 and Ghrelin Attenuate High Glucose/High Lipid-Induced Apoptosis and Senescence of Human Microvascular Endothelial Cells.

BACKGROUND/AIMS: GLP-1 and ghrelin are common appetite-regulating hormones. Both have multiple functions beyond metabolic regulation. However, the effects of GLP-1 and ghrelin on endothelial biology are not fully understood. Here, we investigate the roles of GLP-1 and ghrelin in microvascular endothelial apoptosis and senescence. METHODS: Human microvascular endothelial cells (HMECs) were exposed to high glucose/high lipid (HG/HL) conditions and treated with GLP-1 or ghrelin. Cellular apoptosis, senescence, and mitochondrial function were measured. In addition, the MAPK and Akt signaling pathways were examined. RESULTS: Both GLP-1 and ghrelin treatment decreased the number of TUNEL-positive cells and inhibited caspase-3 and PARP cleavage and mitochondrial dysfunction in HG/HL-exposed HMECs. GLP-1, but not ghrelin decreased the number of ß-galactosidase (ß-gal)-positive cells. Furthermore, GLP-1 and ghrelin inhibited ERK1/2, JNK1/2, and p38 signaling. GLP-1 suppressed Akt signaling, but ghrelin had no effect. Moreover, JNK1/2 and p38 inhibitors, but not ERK1/2 and Akt inhibitors, decreased the number of TUNEL-positive cells. Additionally, only the Akt inhibitor decreased the number of ß-gal-positive cells. CONCLUSION: These results demonstrate that GLP-1 and ghrelin inhibit mitochondrial dysfunction under HG/HL conditions, and suppress endothelial apoptosis via inhibiting JNK1/2 and p38 signaling; moreover, GLP-1 alleviates endothelial senescence via inactivating Akt signaling.

Exploring a common mechanism of alcohol-induced deregulation of RNA Pol III genes in liver and breast cells.

Alcohol intake is associated with numbers of different human cancers, such as hepatocellular carcinoma (HCC) and breast cancer. However, the molecular mechanism remains to be elucidated. RNA polymerase III-dependent genes (Pol III genes) deregulation elevates cellular production of tRNAs and 5S rRNA, resulting in an increase in translational capacity, which promote cell transformation and tumor formation. To explore a common mechanism of alcohol-associated human cancers, we have comparably analyzed that alcohol causes deregulation of Pol III genes in liver and breast cells. Our results reveal that alcohol enhances RNA Pol III gene transcription in both liver and breast cells. The induction of Pol III genes caused by alcohol in ER+ breast cancer lines or liver tumor lines are significantly higher than in their non-tumor cell lines. Alcohol increases cellular levels of Brf1 mRNA and protein, (which depeted) Brf1 is a key transcription factor and specifically regulate Pol III gene activity. Alcohol activates JNK1 to upregulate transcription of Brf1 and Pol III genes, whereas inhibition of JNK1 by SP600125 or its siRNA significantly decreases the induction of these genes. Furthermore, alcohol increases the rates of transformation of liver and breast cells, repressed JNK1 and Brf1 expression decrease transcription of Pol III genes and reduce the rates of colony formation of AML-12 and MCF-10 cells. Together, these studies support the idea that alcohol induces deregulation of Brf1 and RNA Pol III genes in liver and breast cells, which share a common signaling pathway to promote cell transformation. Through the common mechanism, alcohol-induced deregulation of RNA Pol III genes brings about greater phenotypic changes.