Phenylethyl isothiocyanate induces apoptotic signaling via suppressing phosphatase activity against c-Jun N-terminal kinase

Dietary isothiocyanates induce apoptosis in various cancer cell lines through a c-Jun N-terminal kinase (JNK)-dependent mechanism. We found that phenylethyl isothiocyanate (PEITC) was capable of inducing JNK activation and apoptosis in prostate cancer cell lines with distinct p53 statuses. PEITC induced JNK-mediated apoptotic signaling via a different pathway than that used by DNA-damaging agents, because genotoxicresistant LNCaP prostate cancer cells were equally sensitive to PEITC as parental LNCaP cells. PEITC did not induce significant MKK4 or MKK7 activation and did not activate JNK directly, suggesting that JNK and JNK upstream kinases are not primary targets of PEITC. The JNK dephosphorylation and inactivation rates were decreased in cells exposed to PEITC. Expression levels of M3/6, a JNK-specific phosphatase, were down-regulated by PEITC via a proteasome-dependent mechanism. Taken together, our data suggest that PEITC activates JNK through suppression of JNK dephosphorylation and that PEITC may be an alternative therapeutic agent for cancers that are resistant to genotoxic agents. This study also reveals that JNK phosphatases are potential targets for the development of novel cancer therapeutic agents.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Regulation of c-Jun N-terminal kinase activation in hydrogen peroxide induced neurotoxicity

C-Jun N-terminal kinase 1 and 2 (JNK1/2) have been shown to be transiently activated and involved in neurotoxicity. We searched for possible upstream molecules, which are responsible for the regulation of hydrogen peroxide-(H₂O₂) induced JNK1/2 activation and JNK1/2-mediated apoptotic-like cell death in cultured rat cortical neurons. The results showed that JNK1/2 activation (monitored by anti-diphosphorylated JNK1/2 antibody) was largely prevented by elimination of extracellular Ca²⁺ or blockage of NMDA-receptors (NMDA-R), and was weakly but significantly decreased by blockage of L-type voltage-gated calcium channel (L-VGCC); furthermore, JNK1/2 activation was largely prevented by inhibition of Ca²⁺/calmodulin-dependent protein kinase-II (CaMKII) and protein-tyrosine kinases (PTK). We also found that H₂O₂-induced apoptotic-like cell death was partially prevented by elimination of extracellular Ca²⁺, or by inhibition of NMDA-R, L-VGCC, PTK and CaMKII, respectively. The above results suggest that in H₂O₂-induced neurotoxicity, JNK1/2 activation is mainly mediated by NMDA-R and L-VGCC. Consequently, PTK and CaMKII are critical intermediaries in JNK1/2 activation and are mainly responsible for JNK1/2-mediated apoptotic-like cell death.     

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.     

Cell-type-specific activation of c-Jun N-terminal kinase by salicylates

Salicylates inhibit signaling by tumor necrosis factor (TNF), including TNF-induced activation of mitogen-activated protein kinases (MAPKs). On the other hand, we recently showed that in normal human diploid fibroblasts sodium salicylate (NaSal) elicits activation of p38 MAPK but not activation of c-Jun N-terminal kinase (JNK). Here we show that NaSal treatment of COS-1 or HT-29 cells produced a sustained c-Jun N-terminal kinase (JNK) activation. Activation of JNK or p38 MAPK by NaSal (or aspirin) was not due to a nonspecific hyperosmotic effect because much higher molar concentrations of sorbitol or NaCl were required to produce a similar activation. Three structurally unrelated nonsteroidal antiinflammatory drugs (ibuprofen, acetaminophen, and indomethacin) failed to induce significant activation of JNK or p38 MAPK, suggesting that cyclooxygenase inhibition is not the underlying mechanism whereby salicylates induce p38 MAPK and JNK activation. Activation of JNK and p38 MAPKs may be relevant for some antiinflammatory actions of salicylates.     

Hematopoietic progenitor kinase 1 is a component of transforming growth factor beta-induced c-Jun N-terminal kinase signaling cascade

The c-Jun N-terminal kinase (JNK) signaling pathway is involved in transforming growth factor beta (TGF-beta) signaling in a variety of cell systems. We report here that hematopoietic progenitor kinase 1 (HPK1), a novel Ste20-like protein serine/threonine kinase, serves as an upstream mediator for the TGF-beta-activated JNK1 cascade in 293T cells. TGF-beta treatment resulted in a time-dependent activation of HPK1, which was accompanied by similar kinetics of JNK1 activation. The activation of JNK1 by TGF-beta was abrogated by a kinase-defective HPK1 mutant but not by a kinase-defective mutant of kinase homologous to Ste20/Sps1. This result indicates that HPK1 is specifically required for TGF-beta-induced activation of JNK1. We also found that TGF-beta-induced JNK1 activation was blocked by a kinase-defective mutant of TGF-beta-activated kinase 1 (TAK1). In addition, interaction between HPK1 and TAK1 was observed in transient transfection assays, and this interaction was enhanced by TGF-beta treatment. Both stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK) and mitogen-activated protein kinase kinase 7 (MKK7) are immediate upstream activators of JNK1. Although SEK and MKK7 acted downstream of TAK1, only a kinase-defective SEK mutant blocked TGF-beta-induced activation of JNK1, indicating that the TGF-beta signal is relayed solely through SEK, but not MKK7, in vivo. Furthermore, TGF-beta-induced activating protein 1 activation was blocked by a HPK1 mutant, as well as by TAK1 and SEK mutants. Taken together, these studies establish a potential cascade of TGF-beta-activated interacting kinases beginning with HPK1, a Ste20 homolog, and ending in JNK1 activation: HPK1 --> TAK1 --> SEK --> JNK1.     

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.     

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.     

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna^H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna^H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.     

Increased constitutive c-Jun N-terminal kinase signaling in mice lacking glutathione S-transferase Pi

Glutathione S-transferase Pi (GSTP) detoxifies electrophiles by catalyzing their conjugation with reduced glutathione. A second function of this protein in cell defense has recently been proposed that is related to its ability to interact with c-Jun N-terminal kinase (JNK). The present study aimed to determine whether this interaction results in increased constitutive JNK activity in the absence of GSTP in GstP1/P2(-/-) mice and whether such a phenomenon leads to the up-regulation of genes that are relevant to cell defense. We found a significant increase in constitutive JNK activity in the liver and lung of GstP1/P2-/- compared with GstP1/P2(+/+) mice. The greatest increase in constitutive JNK activity was observed in null liver and was accompanied by a significant increase in activator protein-1 DNA binding activity (8-fold) and in the mRNA levels for the antioxidant protein heme oxygenase-1 compared with wild type. Furthermore UDP-glucuronosyltransferase 1A6 mRNA levels were significantly higher in the livers of GstP1/P2(-/-) compared with GstP1/P2(+/+) mice, which correlated to a 2-fold increase in constitutive activity both in vitro and in vivo. There was no difference in the gene expression of other UDP-glucuronosyltransferase isoforms, manganese superoxide dismutase, microsomal epoxide hydrolase, or GSTA1 between GstP1/P2(-/-) and GstP1/P2(+/+) mice. Additionally there was no phenotypic difference in the induction of heme oxygenase-1 mRNA after acetaminophen administration. This study not only demonstrates the role of GSTP as a direct inhibitor of JNK in vivo but also its role in regulating the constitutive expression of specific downstream molecular targets of the JNK signaling pathway.     

c-Jun N-terminal kinase pathways in diabetes

Type 2 diabetes develops from insulin resistance and has become a worldwide epidemic. The c-Jun N-terminal kinases have been considered as signaling molecules linking inflammation and insulin resistance. Genetic disruption of c-Jun N-terminal kinase-1 gene prevents the development of insulin resistance in obese and diabetic mice. Inhibition of c-Jun N-terminal kinases by a small cell-permeable peptide improves insulin sensitivity in mice. Hepatic inhibition of c-Jun N-terminal kinases using a dominant-negative protein or knockdown of c-Jun N-terminal kinase-1 gene by RNA interference reduces blood glucose and insulin levels and enhances hepatic insulin signaling in mice. Recent evidence demonstrates that the hepatic c-Jun N-terminal kinase pathway plays an important role in lipid and lipoprotein homeostasis in mice. This review discusses recent advances in our understanding of the role of c-Jun N-terminal kinase pathway in metabolic control and its potential as a target for the treatment of type 2 diabetes.     

The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the mitochondrial-induced apoptotic pathway

The signaling mechanism by which JNK affects mitochondria is critical to initiate apoptosis. Here we show that the absence of JNK provides a partial resistance to the toxic effect of the heavy metal cadmium. Both wild type and jnk−/− fibroblasts undergoing death exhibit cytosolic cytochrome c but, unlike wild type cells, the JNK-deficient fibroblasts do not display increased caspase activity and DNA fragmentation. The absence of apoptotic death correlates with a specific defect in activation of Bax. We conclude that JNK-dependent regulation of Bax is essential to mediate the apoptotic release of cytochrome c regardless of Bid and Bim activation.     

Regulation of c-Myc through phosphorylation at Ser-62 and Ser-71 by c-Jun N-terminal kinase.

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.     

Long-acting kappa opioid antagonists disrupt receptor signaling and produce noncompetitive effects by activating c-Jun N-terminal kinase

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [(3)H]U69,593 to KOR showed that K(d) and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.     

Endothelin promotes neurite elongation by a mechanism dependent on c-Jun N-terminal kinase

Endothelin (ET), which is known as a vasoconstrictive peptide, is associated with a lot of biological functions. Although endothelin receptors are expressed in the central nervous system (CNS), little is known about the effects of endothelin on neuronal function. In this study, we reported that endothelins elongate cortical neurites via the endothelin A receptor. All the endothelin isoforms tested, endothelin-1, endothelin-2, and endothelin-3, promoted neurite elongation. ET-1-induced neurite elongation was specifically inhibited by treatment with BQ123, an antagonist for the endothelin A receptor. In addition, inhibition of ET-1-induced c-Jun N-terminal kinase (JNK) activation by treatment with SP600125, a JNK inhibitor, also prevented the ET-1-mediated promotion of neurite elongation. Thus, endothelin induces cortical neurite elongation through the endothelin A receptor by a mechanism dependent on JNK.     

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) (Ser(312) in human IRS1). Here we show that JNK mediated feedback inhibition of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity required phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin was inhibited by a cell-permeable peptide that disrupted the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 was not required for its activation by insulin, whereas direct binding was required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1.