Inhibition Of c-Jun Phosphorylation Research Articles

Structure Optimization of c-Jun N-terminal Kinase 1 Inhibitors for Treating Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease with an elusive etiology. Aberrant activation of c-Jun N-terminal kinase 1 (JNK1) has been implicated in its pathogenesis. Through a combination of structure-based drug design and structure-activity relationship (SAR) optimization, a series of pyrimidine-2,4-diamine scaffold derivatives have been developed as potent JNK1 inhibitors. Compound E1 was identified with low nanomolar JNK1 inhibitory potency (IC50 = 2.7 nM). The introduction of a dimethylamine side chain has significantly enhanced the ability of E1 to inhibit c-Jun phosphorylation, surpassing the clinical candidate CC-90001. Molecular dynamics simulations revealed a binding free energy of -50.46 kcal/mol for E1. Moreover, E1 displayed satisfactory pharmacokinetic properties, with a bioavailability of 69% in rats. Furthermore, compound E1 exerted significant antifibrotic effects in a bleomycin-induced IPF mouse model and prevented a TGF-β-induced epithelial-to-mesenchymal transition in vitro. These findings position E1 as a promising lead for further drug development targeting IPF.

Pref-1 induced lung fibroblast differentiation by hypoxia through integrin α5β1/ERK/AP-1 cascade

Chronic obstructive asthma is characterized by airway fibrosis. Hypoxia and connective tissue growth factor (CTGF) play important roles in airway fibrosis. Preadipocyte factor-1 (Pref-1) participates in adipocyte differentiation and liver fibrosis. Herein, we investigated the role of Pref-1 in airway fibrosis in chronic obstructive asthma. We found that Pref-1 was overexpressed in lung tissues from chronic obstructive asthma patients compared to normal subjects. Extracellular matrix proteins were inhibited by Pref-1 small interfering (si)RNA in airway fibroblasts from chronic obstructive asthma patients. Furthermore, ovalbumin induced prominent Pref-1 expression and fibronectin coexpression. Hypoxia induced Pref-1 upregulation and its release into medium of WI-38 cells. Hypoxia-induced CTGF expression was inhibited by Pref-1 siRNA. We also found that Pref-1-stimulated fibrotic protein expressions were reduced by ATN-161, curcumin, U0126, and c-Jun siRNA in WI-38. Furthermore, ATN161 inhibited Pref-1-induced ERK phosphorylation, and ITGA5 siRNA inhibited c-Jun phosphorylation. Moreover, expression of CTGF, Fibronectin, α-SMA, and ERK and c-Jun phosphorylation were all increased in fibroblasts from patients with chronic obstructive asthma. Taken together, these results suggest that Pref-1 participates in airway fibrosis and hypoxia-induced CTGF expression via the integrin receptor α5β1/ERK/AP-1 pathway.

Abstract 2383: EGFR expression increases following decline in activity of N-acetylgalactosamine 4-sulfatase (ARSB) and SHP2 (PTPN11) and increases in chondroitin 4-sulfate and JNK in prostate

Abstract The enzyme arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) removes 4-sulfate groups from the non-reducing end of dermatan sulfate and chondroitin 4-sulfate (C4S) and is required for degradation of these sulfated glycosaminoglycans. Decline in ARSB and the resultant increase in C4S was previously reported to inhibit the activity of the enzyme SHP2 (PTPN11), a ubiquitous, non-receptor tyrosine phosphatase, and to lead to transcriptional events mediated by the sustained phosphorylation of phospho-ERK1,2 and phospho-p38. Previous experiments had shown that decline in ARSB was associated with increased expression of the epidermal growth factor receptor (EGFR; Her1/ErbB1) in human prostate malignant tissue and in human prostate cells, but the mechanism for this increase was unknown. The current experiments were performed to determine how decline in ARSB leads to increased expression of EGFR. Experiments were performed in human prostate stem and epithelial cells and normal and malignant human prostate tissues. Measurements included: EGFR promoter activity, chromatin immunoprecipitation assay, and binding studies of chondroitin sulfates with SHP2. Materials used included: SR11302, an inhibitor of c-fos binding with nuclear DNA; c-Jun peptide that acts as a c-Jun mimetic and inhibits c-Jun phosphorylation by JNK; JNK-selective inhibitor peptide that inhibits JNK phosphorylation; SHP2 dominant negative and constitutively active DNA constructs; PHPS1, an inhibitor of SHP2; ARSB siRNA, and galectin-3 siRNA. Increased EGFR promoter activation and EGFR expression followed decline in ARSB, decline in activity of SHP2 (PTPN11), enhanced activity of c-Jun N-terminal kinase (JNK), increased nuclear DNA binding of c-Jun and c-Fos, and EGFR promoter activation. These effects demonstrate an impact of ARSB and chondroitin 4-sulfation on the activation of a critical dual phosphorylation threonine-tyrosine kinase and on the mRNA expression of an important tyrosine kinase receptor in prostate cells. Restoration of ARSB activity may provide a new therapeutic approach to malignancies in which there is activation of EGFR-mediated tyrosine kinase signaling pathways. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne K. Tobacman. EGFR expression increases following decline in activity of N-acetylgalactosamine 4-sulfatase (ARSB) and SHP2 (PTPN11) and increases in chondroitin 4-sulfate and JNK in prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2383.

Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells

PurposeThe exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells.MethodsThe clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively.ResultsUsing publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment.ConclusionOur findings demonstrate that FN expression is regulated via an AP-1–dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

Abstract 4641: Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation

Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the present study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP down-regulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A. Citation Format: Chuanshu Huang, Junlan Zhu, Jingxia Li. Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4641. doi:10.1158/1538-7445.AM2015-4641

Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis

Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure.

Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the cheliensisin a inhibition of cell transformation.

Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A.

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress, Autophagy, and Apoptosis

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

Two-Step Binding of Transcription Factors Causes Sequential Chromatin Structural Changes at the Activated IL-2 Promoter

Most gene promoters have multiple binding sequences for many transcription factors, but the contribution of each of these factors to chromatin remodeling is still unclear. Although we previously found a dynamic change in the arrangement of nucleosome arrays at the Il2 promoter during T cell activation, its timing preceded that of a decrease in nucleosome occupancy at the promoter. In this article, we show that the initial nucleosome rearrangement was temporally correlated with the binding of NFAT1 and AP-1 (Fos/Jun), whereas the second step occurred in parallel with the recruitment of other transcription factors and RNA polymerase II. Pharmacologic inhibitors for activation of NFAT1 or induction of Fos blocked the initial phase in the sequential changes. This step was not affected, however, by inhibition of c-Jun phosphorylation, which instead blocked the binding of the late transcription factors, the recruitment of CREB-binding protein, and the acetylation of histone H3 at lysine 27. Thus, the sequential recruitment of transcription factors appears to facilitate two separate steps in chromatin remodeling at the Il2 locus.

CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1

CSN1 is a component of the COP9 signalosome (CSN), a conserved protein complex with pleiotropic functions in many organs and cell types. CSN regulates ubiquitinproteasome dependent protein degradation via the deneddylation and the associated deubiquitination activities. In addition, CSN associates with protein kinases and modulates cell signaling, particularly the activator protein 1 (AP-1) pathway. We have shown previously that CSN1 suppresses AP-1 transcription activity and inhibits ultraviolet (UV) and serum activation of c-fos expression. Here we show that CSN1 can inhibit phosphorylation of proto-oncogene c-Jun product and repress c-Jun dependent transcription. Further, CSN1 dramatically downregulates ectopic expression of c-Jun N-terminal kinase 1 (JNK1) in cultured cells. The decline in JNK1 is not caused by excessive proteolysis or by 3' UTR-dependent mRNA instability, but by CSN1-dependent repression of one or multiple steps in transcriptional and posttranscriptional mechanisms. Thus, in contrast to CSN5/Jab1, which promotes AP-1 activity, CSN1 displays a negative effect on the AP-1 pathway. Finally, we discuss about the dynamic equilibrium of the CSN complexes in regulation of the AP-1 pathway.

Characterization of a novel JNK (c-Jun N-terminal kinase) inhibitory peptide

An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.

P27 Suppresses Arsenite-induced Hsp27/Hsp70 Expression through Inhibiting JNK2/c-Jun- and HSF-1-dependent Pathways

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.

Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP+), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 microM MPP+, p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12-24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP+ that triggered the proteasome-mediated degradation of p35. MPP+ treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP+-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP+ treatment. These results indicate that a brief MPP+ treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP+-induced neuronal cell death.

The role of c-Jun phosphorylation in EpRE activation of phase II genes

The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.

JNK is activated but does not mediate hippocampal neuronal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.

Tomatidine inhibits iNOS and COX-2 through suppression of NF-κB and JNK pathways in LPS-stimulated mouse macrophages

We use the LPS-stimulated macrophage as a model of inflammation to investigate the anti-inflammatory effects of tomatidine and solasodine, whose structures resemble glucocorticoids. We found that tomatidine exhibited a more potent anti-inflammatory effect than solasodine. Tomatidine could decrease inducible nitric oxide synthase and cyclooxygenase-2 expression through suppression of I-κBα phosphorylation, NF-κB nuclear translocation and JNK activation, which in turn inhibits c-jun phosphorylation and Oct-2 expression. Here, we demonstrate that tomatidine acts as an anti-inflammatory agent by blocking NF-κB and JNK signaling, and may possibly be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.

JunD Protects the Liver from Ischemia/Reperfusion Injury by Dampening AP-1 Transcriptional Activation

The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD-/- mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/- littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases.

Required Roles of Bax and JNKs in Central and Peripheral Nervous System Death of Retinoblastoma-deficient Mice

Retinoblastoma-deficient mice show massive neuronal damage and deficits in both CNS and PNS tissue. Previous work in the field has shown that death is regulated through distinct processes where CNS tissue undergoes death regulated by the tumor suppressor p53 and the apoptosome component, APAF1. Death in the PNS, however, is independent of p53 and reliant on the death protease, caspase 3. In the present study, we more carefully delineated the common and distinct mechanisms of death regulation by examining the stress-activated kinases, JNK2 and 3, the conserved Bcl-2 member Bax, and the relationship among these elements including p53. By use of genetic modeling, we show that death in various regions of the CNS and DRGs of the PNS is reliant on Bax. In the CNS, Bax acts downstream of p53. The relevance of the JNKs is more complex, however. Surprisingly, JNK3 deficiency by itself does not inhibit c-Jun phosphorylation and instead, aggravates death in both CNS and PNS tissue. However, JNK2/3 double deficiency blocks death due to Rb loss in both the PNS and CNS. Importantly, the relationships between JNKs, p53, and Bax exhibit regional differences. In the medulla region of the hindbrain in the CNS, JNK2/3 deficiency blocks p53 activation. Moreover, Bax deficiency does not affect c-Jun phosphorylation. This indicates that a JNK-p53-Bax pathway is central in the hindbrain. However, in the diencephalon regions of the forebrain (thalamus), Bax deficiency blocks c-Jun activation, indicating that a Bax-JNK pathway of death is more relevant. In the DRGs of the PNS, a third pathway is present. In this case, a JNK-Bax pathway, independent of p53, regulates damage. Accordingly, our results show that a death regulator Bax is common to death in both PNS and CNS tissue. However, it is regulated by or itself regulates different effectors including the JNKs and p53 depending upon the specific region of the nervous system.

P38α Antagonizes p38γ Activity through c-Jun-dependent Ubiquitin-proteasome Pathways in Regulating Ras Transformation and Stress Response

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.