JNK-dependent Signaling Research Articles

Crystallographic mining driven computer-guided approach to identify the ASK1 inhibitor likely to perturb the catalytic region

The pathological levels of reactive oxygen species (ROS) and oxidative stress has been recognized as a critical driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. ASK1 inhibitors are reported to have the potential to treat clinically important inflammatory pathologies including liver, pulmonary and renal disorders. In view of its biological and pathological significance, inhibition of ASK1 with small molecules has been pursued as an attractive strategy to combat human diseases such as non-alcoholic steatohepatitis (NASH). Despite several ASK1 inhibitors being developed, the failure in Phase 3 clinical trials of most advanced candidate selonsertib’s, underscores to discover therapeutic agents with diverse chemical moiety. Here, by using structural pharmacophore and enumeration strategy on mining co-crystals of ASK1, different scaffolds were generated to enhance the chemical diversity keeping the critical molecular interaction in the catalytic site intact. A total of 15,772 compounds were generated from diverse chemical scaffolds and were evaluated using a virtual screening pipeline. Based on docking and MM-GBSA scores, a lead candidate, S3C-1-D424 was identified from top hits. A comparative molecular dynamics simulations (MD) of APO, Selonsertib and shortlisted potential candidates combined with pharmacokinetics profiling and thermodynamic analysis, demonstrating their suitability as potential ASK1 inhibitors to explore further for establishment towards hit-to-lead campaign.Communicated by Ramaswamy H. Sarma

Effect of 3-formylchromone derivatives on neuroinflammation reactions and JNK and NF-κB regulatory pathways

BACKGROUND: Neuroinflammation is a significant component of the pathogenesis of cerebral ischemia. The JNK and NF-B signaling pathways play a leading role in the progression of brain tissue inflammation, which may represent a promising target for therapeutic effects.
 AIM: To evaluate the effect of new derivatives of 3-formylchromone on the course of neuroinflammation reactions and the activity of JNK and NF-B translational pathways in brain tissue in rats with cerebral ischemia.
 MATERIALS AND METHODS: Cerebral ischemia was modeled in Wistar rats by irreversible right-sided occlusion of the middle cerebral artery. The studied compounds and the reference (ethylmethylhydroxypyridine succinate) were administered per os at doses of 30 and 100 mg/kg, respectively. After 72 hours of ischemia, changes in the concentration of proinflammatory cytokines were evaluated in the cerebrospinal fluid: IL-6, IL-1, IL-8 and TNF-. The content of JNK and NF-B in brain tissue was determined by enzyme immunoassay.
 RESULTS: The use of the test compounds 3FC1, 3FC2, 3FC4 and 3FC5, as well as the reference medicine, contributed to a decrease in the content of proinflammatory markers in the liquor. At the same time, the most significant decrease was noted when the compound 3FC5 was administered to animals, namely, the concentration of IL-1, IL-6, IL-8 and TNF- was lower relative to similar indicators of the group of animals without treatment by 30.0% (p 0.05); 64.5% (p 0.05); 48.5% (p 0.05) and 56.6% (p 0.05), respectively. The use of the fluorine-containing compound 3FC3 did not significantly affect the course of brain tissue inflammation reactions in rats. Evaluation of changes in the activity of JNK and NF-B showed that the studied substances inhibit the NF-B translational pathway and do not affect JNK, which is probably due to the activation of these signaling pathways and the antioxidant potential of the studied molecules.
 CONCLUSIONS: The use of compounds that are derivatives of 3-formylchromone in conditions of experimental cerebral ischemia contributes to the reduction of neuroinflammation reactions by inhibiting the NF-B pathway, without affecting the activity of the JNK-dependent signaling system. The substance with the highest pharmacological effects is the compound 3FC5, which contains a spatially hindered phenolic hydroxyl in its structure.

TGF-β signaling in myeloproliferative neoplasms contributes to myelofibrosis without disrupting the hematopoietic niche.

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre–targeted MSCs prevented the development of myelofibrosis in both MPLW515L and Jak2V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPLW515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPLW515L was intact in Osx-Cre Smad4fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPLW515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

TGF-β Signaling in Stromal Cells Contributes to Myelofibrosis, but Not Niche Disruption, in Myeloproliferative Neoplasms

Myeloproliferative neoplasms are associated with significant alterations in the bone marrow microenvironment that contribute to disease pathogenesis. The most striking alteration is the development of myelofibrosis, which is characterized by extensive collagen deposition in the bone marrow and is associated with a poor prognosis. Recent evidence suggests that expression of key niche factors, including CXCL12 (stromal derived factor-1, SDF-1) and Kit ligand are reduced in MPNs. This is relevant, since studies by our group and others have shown that deleting these niche factors from stromal cells results in a shift in hematopoiesis from the bone marrow to spleen. Indeed, a prominent feature of MPN is the development of splenomegaly and extramedullary hematopoiesis. There is evidence implicating inflammatory mediators in the development of myelofibrosis. In particular, increased production of TGF-β produced by megakaryocytes and monocytes is found in most patients with MPNs. To assess the role of TGF-β signaling in mesenchymal stromal cells in the bone marrow in the development of myelofibrosis, we generated Osx-Cre; Tgfbr2 f/- mice, in which TGF-β signaling is abrogated in all bone marrow mesenchymal stromal cells (including Lepr + stromal cells), but not endothelial cells or hematopoietic cells. We transplanted MPL W515L transduced hematopoietic stem and progenitor cells (HSPCs) or JAK2 V617F bone marrow into these mice and quantified myelofibrosis using reticulin staining and Collagen 1 and 3 immunostaining. We previously reported that deletion of TGF-β signaling in mesenchymal stromal cells in these mice abrogated the development of myelofibrosis, and we presented evidence that this was mediated by non-canonical JNK-dependent TGF-β signaling. Here, we describe the impact of stromal TGF-β signaling on the bone marrow hematopoietic niche in MPN.MPL W515L transduced HSPCs were transplanted into Osx-Cre; Tgfbr2 f/- mice, and the impact on hematopoietic niche disruption and development of extramedullary hematopoiesis was assessed. In control recipients, transplantation of MPL W515L HSPCs resulted in marked decreases in bone marrow Cxcl12 and Kit ligand expression (Figure 1A-B). Surprisingly, a similar decrease was observed in Osx-Cre; Tgfbr2 f/- recipients. The loss of these key niche factors is predicted to impair hematopoietic niche function in the bone marrow. Consistent with this prediction, total bone marrow cellularity and HSC number were significantly reduced in both control and Osx-Cre; Tgfbr2 f/- recipients (Figure 1C-D). Finally, disruption of the bone marrow niche is often associated with extramedullary hematopoiesis. Indeed, a significant increase in spleen size and spleen HSCs and erythroid progenitors was observed in control recipients (Figure 1E-G). Again, a similar phenotype was observed in Osx-Cre; Tgfbr2 f/- recipients.Collectively, these data show that TGF-β signaling in bone marrow mesenchymal stromal cells is required for the development of myelofibrosis but not hematopoietic niche disruption in MPNs. Thus, these data show for the first time that the signals that induce a fibrogenic program in bone marrow mesenchymal stromal cells are distinct from those that suppress Cxcl12 and Kit ligand expression. Our data show that the fibrogenic program is dependent on non-canonical JNK-dependent TGF-β signaling, while the signals that regulate niche factor expression remain unknown. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pathophysiological Significance of WDR62 and JNK Signaling in Human Diseases.

The c-Jun N-terminal kinase (JNK) is highly evolutionarily conserved and plays important roles in a broad range of physiological and pathological processes. The WD40-repeat protein 62 (WDR62) is a scaffold protein that recruits different components of the JNK signaling pathway to regulate several human diseases including neurological disorders, infertility, and tumorigenesis. Recent studies revealed that WDR62 regulates the process of neural stem cell mitosis and germ cell meiosis through JNK signaling. In this review we summarize the roles of WDR62 and JNK signaling in neuronal and non-neuronal contexts and discuss how JNK-dependent signaling regulates both processes. WDR62 is involved in various human disorders via JNK signaling regulation, and may represent a promising therapeutic strategy for the treatment of related diseases.

Oligomannuronate prevents mitochondrial dysfunction induced by IAPP in RINm5F islet cells by inhibition of JNK activation and cell apoptosis

BackgroundOligomannuronates (OM) are natural products from alginate that is frequently used as food supplement. The aim of this study was to investigate the in vitro protective effects of OM on RINm5F cells against human Islet amyloid polypeptide (IAPP) induced mitochondrial dysfunction, as well as the underlying mechanisms.MethodsIn the present study, we obtained several kinds of OM with different molecular masses, and then we used RINm5F cells as a model to elucidate the involvement of JNK signal pathway in hIAPP-induced mitochondrial dysfunction in pancreatic beta cells, and the protective effects of OM are associated with its ability to attenuate the mitochondrial dysfunction.ResultsOur results demonstrated that human IAPP induced mitochondrial dysfunction, as evidence by loss of ΔΨm and ATP content, and decrease in oxygen consumption and complex activities, was accompanied by JNK activation, changes in the expressions of Bcl-2 and Bax proteins, release of cytochrome c (Cyto-c) and apoptosis inducing factor (AIF) from mitochondria into cytosol. Interestingly, the human IAPP induced damage in RINm5F cells were effectively restored by co-treatment of OM. Moreover, JNK activation was required for the OM mediated changes in RINm5F cells.ConclusionsOM prevented mitochondrial dysfunction induced by human IAPP in RINm5F islet cells through JNK dependent signaling pathways.

Diminished Expression of P-glycoprotein Using Focused Ultrasound Is Associated With JNK-Dependent Signaling Pathway in Cerebral Blood Vessels

MRI-guided focused ultrasound (MRgFUS) combined with microbubbles (MBs) is a promising technology that can facilitate drug delivery through a temporarily disrupted blood-brain barrier (BBB) and induce the down-regulation of P-glycoprotein (P-gp) expression on the blood vessels. Despite the increasing evidence regarding the down-regulation of P-gp expression after MRgFUS BBB disruption (BBBD), its underlying molecular events remain unclear. The aim of this study was to evaluate the underlying mechanism of FUS BBBD-mediated P-gp down-regulation. While our results showed down-regulation of P-gp at 24 h post-BBBD in transcriptional and translational levels, restoration to the normal expression appeared at different time points for transcriptional (72 h) and translational (120 h) levels. In addition, the signaling molecule, JNK, was significantly activated in the cerebral blood vessels at 24 h post-BBBD. Although P-gp levels were significantly decreased, the expression levels of proteins involved in the integrity of blood vessels, such as Glut1, ZO-1 and occludin, were not decreased at 24 h post-BBBD. Our study suggests that the JNK signaling pathway is involved in the regulation of FUS-induced P-gp expression, without affecting vessel integrity, and a detailed regulatory mechanism can provide the basis for clinical application of FUS to the treatment of neurological disease.

JNK Signaling Pathway Involvement in Spinal Cord Neuron Development and Death.

The c-Jun NH2-terminal protein kinase (JNK) is a Janus-faced kinase, which, in the nervous system, plays important roles in a broad range of physiological and pathological processes. Three genes, encoding for 10 JNK isoforms, have been identified: jnk1, jnk2, and jnk3. In the developing spinal cord, JNK proteins control neuronal polarity, axon growth/pathfinding, and programmed cell death; in adulthood they can drive degeneration and regeneration, after pathological insults. Indeed, recent studies have highlighted a role for JNK in motor neuron (MN) diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophy. In this review we discuss how JNK-dependent signaling regulates apparently contradictory functions in the spinal cord, in both the developmental and adult stages. In addition, we examine the evidence that the specific targeting of JNK signaling pathway may represent a promising therapeutic strategy for the treatment of MN diseases.

Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies.

Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Oregonin inhibits inflammation and protects against barrier disruption in intestinal epithelial cells

Background and aimsOregonin, a major diarylheptanoid derivative isolated from Alnus japonica, exerts anti-inflammatory effects; however, little is known about the effect of oregonin in intestinal inflammation. The current study investigated the potential of oregonin for clinical applications in the treatment of inflammatory bowel disease (IBD) and elucidated its underlying molecular mechanisms. MethodsThe anti-inflammatory effect of oregonin in tumor necrosis factor-α (TNF-α)-stimulated human intestinal epithelial HT-29 cells was investigated. In addition, the protective effect of oregonin was determined against disruption of the intestinal barrier in tert-butyl hydroperoxide (t-BH)-stimulated human intestinal epithelial Caco-2 cells. ResultsOregonin suppressed the expression of cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1), IL-8, and IL-1β, and inhibited activation of nuclear factor κB (NF-κB) in HT-29 cells stimulated with TNF-α. Oregonin increased heme oxygenase-1 (HO-1) expression through the ERK1/2 and JNK-dependent signaling pathway, which contributed to the oregonin-mediated suppression of COX-2 expression in the HT-29 cells stimulated with TNF-α. Moreover, oregonin induced AMP-activated protein kinase (AMPK) activation. Knockdown of AMPK abolished the induction of HO-1 protein by oregonin and suppression of oregonin-mediated ICAM-1 and COX-2 expression in the HT-29 cells stimulated with TNF-α. Oregonin prevented the t-BH-induced increase in monolayer permeability through inhibition of the reduction in expression of zonula occludens-1 and occludin in Caco-2 cells. Targeting HO-1 by siRNA transfection attenuated the oregonin-mediated prevention of loss of tight junction proteins and increase in permeability. ConclusionThe findings of this study suggest that oregonin is a potential candidate for treatment of IBD by preventing mucosal inflammation and barrier disruption.

Rational approach to highly potent and selective apoptosis signal-regulating kinase 1 (ASK1) inhibitors

Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar potency in a cellular assay, high selectivity when tested against kinase and broad pharmacology screening panels, and attractive physicochemical properties. The compounds we describe are attractive tool compounds to inform the therapeutic potential of ASK1 inhibition.

Trichostatin A ameliorates renal tubulointerstitial fibrosis through modulation of the JNK-dependent Notch-2 signaling pathway

Renal fibrosis is the final common pathological feature in a variety of chronic kidney disease. Trichostatin A (TSA), a histone deacetylase inhibitor, reportedly attenuates renal fibrosis in various kidney disease models. However, the detailed molecular action of TSA in ameliorating renal fibrotic injury is not yet fully understood. In a cultured renal fibroblastic cell model, we showed that TGF-β1 triggers upregulation of α-SMA and fibronectin, two hallmarks of myofibroblastic activation. During the course of TGF-β1 treatment, activation of Smad2/3, p38, ERK, JNK and Notch-2 was also detected. Under the conditions, administration of TSA significantly decreased TGF-β1-stimulated expression of α-SMA, fibronectin, phospho-JNK, and cleaved Notch-2; however, the levels of phospho-Smad2/3, phospho-p38 and phospho-ERK remained unchanged. Pharmacological inhibition of different signaling pathways and genetic knockdown of Notch-2 further revealed JNK as an upstream effector of Notch-2 in TGF-β1-mediated renal fibrosis. Consistently, we also demonstrated that administration of TSA or a γ-secretase inhibitor RO4929097 in the mouse model of unilateral ureteral obstruction significantly ameliorated renal fibrosis through suppression of the JNK/Notch-2 signaling activation. Taken together, our findings provide further insights into the crosstalk among different signaling pathways in renal fibrosis, and elucidate the molecular action of TSA in attenuating fibrogenesis.

Abstract 11919: A Potent and Selective Covalent Inhibitor of JNK Protects Heart From Both Ischemia/Reperfusion- and Hypertension-induced Heart Failure

Introduction: c-Jun NH 2 -terminal kinases (JNKs) are critical mediators of a variety of detrimental stimuli including ischemia-reperfusion (I/R) and mechanical stress induced by pressure overload. A novel synthesized highly selective JNK inhibitor, JNK-IN-8, can form an ATP site-directed covalent bond with a conserved cysteine residual in all JNKs, therefore, it may be broadly useful as a pharmacological tool for inhibiting JNK-dependent signal transduction that is involved in various pathological conditions. Hypothesis: The potent JNK inhibitor, JNK-IN-8, could act as a cardioprotectant against ischemia/reperfusion (I/R)- or hypertension-induced heart failure. Methods: The effects of JNK-IN-8 were monitored in a mouse I/R models with the left anterior descending coronary artery occluded and subsequently released, and a mouse model of transverse aortic constriction (TAC), in which pressure overload hypertrophy was induced in 4 weeks. Results: JNK-IN-8 treatment significantly reduced myocardial infarct area by 40% in mouse ischemia and reperfusion (I/R) model as compared to the vehicle groups (p<0.01). Interestingly, the immunohistochemistry data showed that JNK-IN-8 treatment attenuated the macrophage infiltration and caspase-3 executed apoptosis signaling in TAC-induced heart failure mice (p<0.01 vs. vehicle groups). Moreover, the staining of wheat germ agglutinin (WGA) and Masson trichrome staining revealed that there are significant decreases in hypertrophy and fibrosis in the hearts of JNK-IN-8 treated TAC-induced heart failure mice. Furthermore, the immunoblotting data demonstrated that JNK-IN-8 treatment selectively attenuated both I/R- and pressure overload-induced JNK and downstream oxidative stress p-SHC (p66), inflammatory NF-kappaB signaling pathways, while the levels of other mitogen protein kinase family signals (p-p38 and p-ERK) remained unchanged, indicating the specificity of JNK-IN-8 on JNK signaling in the heart. Conclusions: JNK-IN-8 reduces myocardial infarction caused by ischemia and reperfusion, and attenuates pressure overload-induced inflammation, apoptosis, hypertrophy and fibrosis. Thus JNK-IN-8 is a potential therapeutic drug for I/R- and pressure overload-induced heart failure.

Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes

Objectives: The aim of this study was to investigate the effect and potential mechanism of Cysteine-rich 61 (Cyr61) on stimulating MMP-3 expression by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients.Methods: Primarily cultured RA FLS were treated with exogenous Cyr61 protein or Cyr61-siRNA, then, MMP-3 expression was analyzed by real-time PCR, western blotting and ELISA. Signal transduction pathways in Cyr61-induced MMP-3 production were examined by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as control and MMP-3 in the joint was detected by IHC, real-time PCR and western blotting.Results: High expressed MMP-3 and Cyr61 were positively correlated in RA ST; Cyr61 stimulated MMP-3 production in FLS of RA patients in an IL-1β and TNF-α independent manner. Cyr61 induced MMP-3 could further enhance the invasive ability of RA FLS. Mechanistically, we found that Cyr61 promoted MMP-3 production via the P38, JNK-dependent AP-1 signaling pathway. Blockage of Cyr61 function with monoclonal antibody could decrease MMP-3 expression in the joints of CIA mice.Conclusion: This study provides new evidence that Cyr61 participates in RA pathogenesis not only as a pro-inflammatory factor but also plays a key role in bone erosion via promoting MMP-3 expression. We suggest that targeting of Cyr61 may represent a potential strategy in RA treatment.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states.

GADD45α modulates curcumin sensitivity through c-Abl- and JNK-dependent signaling pathways in a mismatch repair-dependent manner.

Colorectal cancer is a critical health concern because of its incidence as the third most prevalent cancer in the world. Currently, 5-fluorouracil (5-FU), 6-thioguanine, and certain other genotoxic agents are mainstays of treatment; however, patients often die due to emergence of resistant population. Curcumin, a bioactive compound derived from the dietary turmeric (Curcuma longa) is an effective anticancer, anti-inflammatory, and antioxidant agent. Previously, we reported that human colorectal cancer cell lines compromised for mismatch repair (MMR) function exhibit heightened sensitivity to curcumin due to sustained curcumin-induced unrepaired DNA damage compared to proficient population counterparts. In this report, we show that the protein levels of gadd45α, whose transcript levels are increased during DNA damage and stress signals, are upregulated following curcumin treatment in a dose- and time-dependent manner. We further observed that cells compromised for Mlh1 function (HCT116 + Ch2) displayed ~twofold increased GADD45α upregulation compared to similarly treated proficient counterparts (HCT116 + Ch3). Similarly, suppression of Mlh1 using ShRNA increased GADD45α upregulation upon curcumin treatment. On the other hand, suppression of GADD45α using SiRNA-blocked curcumin-induced cell death induction in Mlh1-deficient cells. Moreover, inhibition of Abl through ST571 treatment and its downstream effector JNK through SP600125 treatment blocked GADD45α upregulation and cell death triggered by curcumin. Collective results lead us to conclude that GADD45α modulates curcumin sensitivity through activation of c-Abl > JNK signaling in a mismatch repair-dependent manner.

Impaired dynamin 2 function leads to increased AP-1 transcriptional activity through the JNK/c-Jun pathway

Activation of AP-1 transcription factors, composed of the Jun and Fos proteins, regulates cellular fates, such as proliferation, differentiation or apoptosis. Among other stimuli, the AP-1 pathway can be initiated by extracellular ligands, such as growth factors or cytokines, which undergo internalization in complex with their receptors. Endocytosis has been implicated in the regulation of several signaling pathways; however its possible impact on AP-1 signaling remains unknown. Here we show that inhibition of dynamin 2 (Dyn2), a major regulator of endocytic internalization, strongly stimulates the AP-1 pathway. Specifically, expression of a dominant-negative Dyn2 K44A mutant increases the total levels of c-Jun, its phosphorylation on Ser63/73 and transcription of AP-1 target genes. Interestingly, DNM2 mutations implicated in human neurological disorders exhibit similar effects on AP-1 signaling. Mechanistically, Dyn2 K44A induces AP-1 by increasing phosphorylation of several receptor tyrosine kinases. Their activation is required to initiate a Src- and JNK-dependent signaling cascade converging on c-Jun and stimulating expression of AP-1 target genes. Cumulatively, our data uncover a link between the Dyn2 function and JNK signaling which leads to AP-1 induction.

Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 suppress chronic cerebral hypoperfusion-induced neuronal apoptosis by inhibiting c-Jun N-terminal kinase signaling

The endocannabinoid system (ECS) has therapeutic potential for treating chronic cerebral hypoperfusion (CCH)-induced cerebral diseases. This study investigated the protective effects of two ECS compounds, cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase inhibitor URB597 (URB) on CCH-induced neuronal apoptosis in vivo. CCH was induced in male Sprague–Dawley rats by bilateral common carotid artery occlusion (BCCAo); the rats were then treated with WIN or URB for 12weeks and their spatial learning and memory abilities were assessed using the Morris water maze. Changes in neuronal number were examined by labeling neurons with an antibody against the neuronal nuclei antigen, and apoptosis of cortical and hippocampal CA1 neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The expression of B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax), and activated caspase-3 as well as mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, phosphorylated (p-)ERK, p-JNK, and p-P38 was examined by Western blotting. Rats treated with WIN or URB showed better learning and memory performance than controls. The neuroprotective effects of URB were greater than those of WIN, and co-administration of WIN and URB had a synergistic effect. In addition, WIN and URB blocked JNK phosphorylation as well as the decrease in Bcl-2/Bax ratio and caspase-3 activation induced by CCH, implying that these agents modulate neuronal survival. Moreover, the selective JNK inhibitor SP600125 improved mitochondrial membrane dysfunction and blocked neuronal apoptosis induced by JNK-dependent Bcl-2 signaling. WIN and URB enhanced the effects of SP600125, implying that they may exert anti-apoptotic effects in part by inhibiting a non-nuclear JNK pathway. These findings indicate that WIN and URB promote neuronal survival and may potentially be used to protect neurons against chronic ischemic insults.

Myrosinase-treated glucoerucin is a potent inducer of the Nrf2 target gene heme oxygenase 1 — studies in cultured HT-29 cells and mice

In this study, the effect of myrosinase-treated glucoerucin (GER+MYR), which releases the isothiocyanate (ITC) erucin, on heme oxygenase 1 (HO-1) gene expression and Nrf2 signaling was investigated in vitro in cultured cells and in vivo in mice. Treatment of HT-29 cells with GER+MYR resulted in a significant increase in the mRNA and protein levels of nuclear Nrf2 and HO-1. GER+MYR was more potent at enhancing the nuclear Nrf2 levels than were the following myrosinase-treated glucosinolates: sinigrin, glucoraphanin and gluconasturtiin, which are the precursors of allyl-ITC, R-sulforaphane and 2-phenylethyl ITC, respectively. GER+MYR also significantly induced HO-1 gene expression in the mouse intestinal mucosae and liver but not in the brain. Mechanistic studies suggest that GER+MYR induces Nrf2 via ERK1/2-, p38- and JNK-dependent signal transduction pathways. The GER+MYR-mediated increase in HO-1 expression is primarily attributable to p38 signaling.

Abstract 13262: A Potent and Selective Covalent Inhibitor of JNK Protects Heart from both Ischemia/Reperfusion- and Hypertension-induced Heart Failure

Introduction: c-Jun NH 2 -terminal kinases (JNKs) are critical mediators of a variety of detrimental stimuli including ischemia-reperfusion (I/R) and mechanical stress induced by pressure overload. Recently, we reported that a novel synthesized highly selective JNK inhibitor, JNK-IN-8, can form an ATP site-directed covalent bond with a conserved cysteine residual in all JNKs. Therefore, JNK-IN-8 may be broadly useful as a pharmacological tool for inhibiting JNK-dependent signal transduction that is involved in various pathological conditions. Hypothesis: The potent JNK inhibitor, JNK-IN-8, could act as a cardioprotectant against ischemia/reperfusion (I/R)- or hypertension-induced heart failure. Methods: The effects of JNK-IN-8 were monitored in a mouse I/R models with the left anterior descending coronary artery (LAD) occluded and subsequently released, and a mouse model of trans-aortic constriction (TAC), in which pressure overload hypertrophy was induced in 2 weeks. Results: JNK-IN-8 significantly reduced myocardial infarct area by 40% in I/R model relative to the vehicle group (p<0.01). Interestingly, the immunohistochemistry data showed that JNK-IN-8 treatment attenuated macrophage infiltration and caspase-3 executed apoptosis in TAC mice (p<0.01 vs. vehicle group). Moreover, the staining of wheat germ agglutinin (WGA) and Masson trichrome revealed a decrease in hypertrophy and fibrosis in the TAC hearts of JNK-IN-8 treated group (p<0.05 vs. vehicle group). The immunoblotting data demonstrated that JNK-IN-8 selectively attenuated both I/R- and pressure overload-induced cardiac JNK and downstream p-p66 shc signaling pathways, while the levels of other family signals (p-p38 and p-ERK) remained unchanged, further indicating the specificity of JNK-IN-8 on JNK signaling in the heart. Conclusions: JNK-IN-8 can reduce I/R-injury induced myocardial infarction size, and attenuate pressure overload induced inflammation, apoptosis, hypertrophy and fibrosis. Thus JNK-IN-8 is a potential therapeutic drug for both I/R injury and pressure overload induced hypertrophy.