Upstream Activator Of JNK Research Articles

Dual Mkk4 and Mkk7 Gene Deletion in Adult Mouse Causes an Impairment of Hippocampal Immature Granule Cells.

(1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase involved in regulating physiological processes in the central nervous system. However, the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/−), Mkk4flox/flox, Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old mice induced the gene deletion (Actin-CreERT2 (Cre+/−), Mkk4∆/∆, Mkk7∆/∆ genotype), which was verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment position of immature hippocampal neurons together with alterations in their dendritic architecture pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: All these data supported that the MKK4/MKK7–JNK pathway has a role in adult neurogenic activity.

LECT2 as a hepatokine links liver steatosis to inflammation via activating tissue macrophages in NASH

It remains unclear how hepatic steatosis links to inflammation. Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that senses fat in the liver and is upregulated prior to weight gain. The aim of this study was to investigate the significance of LECT2 in the development of nonalcoholic steatohepatitis (NASH). In human liver biopsy samples, elevated LECT2 mRNA levels were positively correlated with body mass index (BMI) and increased in patients who have steatosis and inflammation in the liver. LECT2 mRNA levels were also positively correlated with the mRNA levels of the inflammatory genes CCR2 and TLR4. In C57BL/6J mice fed with a high-fat diet, mRNA levels of the inflammatory cytokines Tnfa and Nos2 were significantly lower in Lect2 KO mice. In flow cytometry analyses, the number of M1-like macrophages and M1/M2 ratio were significantly lower in Lect2 KO mice than in WT mice. In KUP5, mouse kupffer cell line, LECT2 selectively enhanced the LPS-induced phosphorylation of JNK, but not that of ERK and p38. Consistently, LECT2 enhanced the LPS-induced phosphorylation of MKK4 and TAB2, upstream activators of JNK. Hepatic expression of LECT2 is upregulated in association with the inflammatory signature in human liver tissues. The elevation of LECT2 shifts liver residual macrophage to the M1-like phenotype, and contributes to the development of liver inflammation. These findings shed light on the hepatokine LECT2 as a potential therapeutic target that can dissociate liver steatosis from inflammation.

PKCδ contributes to oxidative stress-induced apoptosis in porcine ovarian granulosa cells via activating JNK

Oxidative stress-induced apoptosis of granulosa cells (GCs) is believed to be an important cause of follicular atresia. Our previous work showed that the c-Jun N-terminal kinase (also known as JNK) might promote apoptosis in GCs during oxidative stress. The aim of this study was to investigate the upstream signaling required for JNK-mediated GCs apoptosis during oxidative stress. Since PKCδ and ASK1 have been suggested to regulate JNK activity in some types of cells, we hypothesized that PKCδ and ASK1 might contribute to JNK-dependent apoptosis in GCs suffering oxidative stimulation. To test this assumption, porcine GCs obtained from healthy follicles were treated with H2O2 alone, or together with inhibitors against PKCδ and JNK, and then collected for cell viability assay, TUNEL staining, immunoprecipitation, western blotting, or JNK activity detection in vitro. The current results showed that the cell viability loss, DNA fragmentation, morphological shrinkage, and nuclear condensation in H2O2-treated porcine GCs was correlated with enhanced activation of JNK. Although ASK1 was supposed to be a JNK activator, we found no definite role of ASK1 in JNK-induced GCs apoptosis during oxidative stress. Further investigations revealed that H2O2-mediated PKCδ activation was required for the apoptotic death of porcine GCs. Particularly, the pro-apoptotic effects of PKCδ on porcine GCs might be achieved by activating the mitochondrial pathway. Importantly, we found that p-PKCδ acts as an upstream activator of JNK in H2O2-treated porcine GCs. However, JNK has no regulatory effect on PKCδ activity. Taken together, our findings provided a novel model of GCs apoptosis involving the activation of PKCδ/JNK/mitochondrial apoptosis axis during oxidative stress.

Costunolide enhances doxorubicin-induced apoptosis in prostate cancer cells via activated mitogen-activated protein kinases and generation of reactive oxygen species.

The management of castration-resistant prostate cancer (CRPC) is challenging, attributable to a lack of efficacious therapies. Chemotherapy is one of the most important treatments for CRPC. Doxorubicin has been extensively used in many different tumors and is often combined with other drugs to enhance effects and reduce toxicity. Costunolide is a natural sesquiterpene lactone with anti-cancer properties. In this study, we first demonstrated that the combination of costunolide and doxorubicin induced apoptosis significantly more than either drug alone in prostate cancer cell lines. Costunolide combined with doxorubicin induced mitochondria-mediated apoptosis through a loss of mitochondrial membrane potential and modulation of Bcl-2 family proteins. We found that this drug combination significantly increased the production of reactive oxygen species (ROS), as well as phosphorylation of c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, which play upstream roles in mitochondria-mediated apoptosis. Further studies showed that N-acetyl cysteine blocked JNK and p38 phosphorylation, suggesting that ROS were upstream activators of JNK and p38. However, a JNK inhibitor, but not a p38 inhibitor, blocked the increase in ROS observed in cells treated with a combination of costunolide and doxorubicin, suggesting that ROS and JNK could activate each other. In vivo, inhibition of tumor growth and induction of apoptosis were greater in mice treated with the costunolide and doxorubicin combination than in mice treated with either drug alone, without an increase in toxicity. Therefore, we suggested that costunolide in combination with doxorubicin was a new potential chemotherapeutic strategy for treating prostate cancer.

JNK does not regulate meiotic progression in Xenopus oocytes: The strange case of pJNK and pERK

Xenopus ERK2, also known as Xp42 MAPK, is activated by progesterone and regulates meiotic progression in the oocytes through activation of the phosphatase Cdc25C and inhibition of the protein kinase Myt1, thus promoting dephosphorylation and activation of cyclinB/Cdc2 (MPF). Indeed, it has been reported that stress protein kinases p38 and JNK are activated during meiotic progression and, more specifically, that p38γ regulates meiosis through activation of Cdc25C. However, the role of JNK in meiotic progression is not so clear, and despite a 42kDa protein is detected with pJNK antibodies (XpJNK-p42), the specific isoform activated by progesterone has not been characterized in detail. The serine/threonine kinase MEKK1, an upstream activator of JNK and p38, is activated during stress conditions and regulates apoptosis in different cell types. Here we show that ectopic expression of a constitutively active MEKK1 in Xenopus oocytes induces phosphorylation of p38, JNK and ERK and accelerates meiotic progression induced by progesterone. Inhibition of each individual pathway reduces the acceleration of meiosis induced by MEKK1. However, constitutively active MEKK1 induces phosphorylation of two JNK isoforms (p40 and p49, corresponding to JNK1–1 and JNK1–2 respectively) distinct to the p42 protein detected with pJNK antibodies during meiotic progression (XpJNK-p42). Moreover, a constitutively active MKK7, which specifically activates the JNK signaling pathway and induces phosphorylation of the p40 and p49 isoforms, does not accelerate meiotic progression. Immunoprecipitation of the p42 protein with pJNK antibodies and subsequent analysis by mass spectrometry shows that XpJNK-p42 is, in fact, pERK2. Ectopic expression of ERK2 in oocytes treated with progesterone or hyperosmotic shock indicates that ERK2 is phosphorylated in both conditions but is only detected with pJNK antibodies in progesterone-treated oocytes. In addition, mature oocytes only present a moderate increase of Jun kinase activity, which is not inhibited by SP600125. In conclusion, JNKs are not activated during meiotic progression and XpJNK-p42 is a post-translational modification of pERK induced by progesterone.

Identification and characterization of MKK7 as an upstream activator of JNK in Litopenaeus vannamei.

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

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury.

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen–glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.

Neuroprotection by MKK7 inhibition in excitotoxicity

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads neurons to death. One key molecule of this pathway is c-Jun-N-terminal kinase (JNK), a MAP kinase, which plays both physiological and pathological roles in neurons. We have previously shown that JNK blockade by specific cell permeable peptide inhibitors significantly reduces infarct size and neuronal death. On the other hand, JNK inhibition may have detrimental side effects due to blockade of its physiological function. Here we have designed a new inhibitor, which blocks MKK7, an upstream activator of JNK, which mediates its pathological activation. This inhibitor was designed taking advantage of the growth arrest and DNA damage inducible 45β (GADD45β) ability to bind MKK7, optimizing the essential domain of GADD45β and linking it with a spacer to TAT peptide sequence to penetrate cells. This inhibitor significantly reduces neuronal death in two in vitro models of excitotoxic cell death, one induced by NMDA exposure and the other by oxygen glucose deprivation. We tested the MKK7 inhibitor in vivo, in two models of permanent ischemia, the one obtained by electrocoagulation, and the other by thromboembolic occlusion of the Middle Cerebral Artery. In both models, it blocked MKK7 activation and provided significant protection, significantly reducing the infarct size when injected 30’ before the lesion. In the electrocoagulation model, we also tested the efficacy of the peptide when injected 6h after lesion, obtaining similar protection. Therefore, we showed that it is possible to prevent JNK activation in excitotoxicity by specific inhibition of MKK7, preserving the physiological role of JNK driven by MKK4. Targeting MKK7 could represent a novel therapeutic strategy for several diseases involving JNK activation.

Abstract 2872: Induction of autophagy in hypoxic HT29 cells requires functional JNK1

Abstract BACKGROUND: Hypoxic HT29 colon cancer cell lines exhibit higher sensitivity to oxaliplatin when SEK1 function is impaired and higher resistance when MKK7 is down regulated (which in turn, cause more profound inhibition of JNK activation). We observed differential effects of JNK1/JNK2 modulation on oxaliplatin sensitivity of hypoxic HT29 cells to oxaliplatin treatment that implied a pro-survival function for JNK1. Further, we have shown that both oxaliplatin- and hypoxia-induced autophagy in these cells is diminished in the presence of JNK inhibitor SP600125, and that the cells are sensitized thereby. PURPOSE: Here we investigate further how modulation of signaling through JNK influences autophagy in hypoxic HT29 cells treated with oxaliplatin, and expand the cellular model to additional colon cancer cell lines. We created a panel of HT29-derived cell lines stably expressing dominant negative constructs for SEK1 (HTS13), MKK7 (HTM9), JNK1 (HTJ1.3) and JNK2 (HTJ2.2). We also utilized 6 colon cancer cell lines (HCT116, LoVo, RKO1, SW480, DLD1 and HCT15) in which a dominant negative construct for JNK1 was introduced by viral delivery. RESULTS: Our data demonstrate that in HTJ1.3 cells, hypoxic induction of autophagy is impaired. Silencing of JNK2 in these cells by viral delivery of shRNA does not affect either autophagy induction, or sensitivity to oxaliplatin. Downregulation of up-stream activators of JNK, as in the dnSEK1 or dnMKK7 derivatives, similarly does not affect autophagy induction, while still demonstrating differential effects on activation of JNK isoforms and its major target, c-Jun. Finally, we show that colon cancer cell lines differ in their ability initiate autophagy under hypoxia. Correspondingly, the introduction of dominant negative JNK1 causes some enhancement of oxaliplatin cytotoxicity in LoVo, RKO1 and HCT15 cell lines, which all demonstrate a greater propensity to autophagy under these circumstances. CONCLUSIONS: Our data demonstrate that JNK1 plays a central role in autophagy induction under hypoxic conditions. Knockdown of JNK1 renders HT29 cells more sensitive to hypoxia due to impaired autophagy induction, and JNK2 is unable to substitute for JNK1 under these conditions. Our results also imply involvement of alternative JNK1 activators in autophagy induction by hypoxia. Finally, induction of autophagy and enhancement of oxaliplatin cytotoxicity, by this mechanism, appear to be model-specific in colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2872. doi:10.1158/1538-7445.AM2011-2872

Gadd45β forms a Homodimeric Complex that Binds Tightly to MKK7

Gadd45α, β, and γ proteins, also known as growth arrest and DNA damage-inducible factors, have a number of cellular functions, including cell-cycle regulation and propagation of signals produced by a variety of cellular stimuli, maintaining genomic stability and apoptosis. Furthermore, Gadd45β has been indicated as a major player in the endogenous NF-κB-mediated resistance to apoptosis in a variety of cell lines. In fibroblasts this mechanism involves the inactivation of MKK7, the upstream activator of JNK, by direct binding within the kinase ATP pocket. On the basis of a number of experimental data, the structures of Gadd45β and the Gadd45β-MKK7 complex have been predicted recently and data show that interactions are mediated by acidic loops 1 and 2, and helices 3 and 4 of Gadd45β. Here, we provide further evidence that Gadd45β is a prevailingly α-helical protein and that in solution it is able to form non covalent dimers but not higher-order oligomers, in contrast to what has been reported for the homologous Gadd45α. We show that the contact region between the two monomers is comprised of the predicted helix 1 (residues Q17–Q33) and helix 5 (residues K131–R146) of the protein, which appear to be antiparallel and to form a large dimerisation surface not involved in MKK7 recognition. The results suggest the occurrence of a large complex containing at least an MKK7-Gadd45β:Gadd45β-MKK7 tetrameric unit whose complexity could be further increased by the dimeric nature of the isolated MKK7.

C-Jun N-terminal Kinase Is Necessary for Platelet-derived Growth Factor-mediated Chemotaxis in Primary Fibroblasts

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

C-Jun N-Terminal Kinase (JNK) Regulation of iNOS Expression in Glial Cells: Predominant Role of JNK1 Isoform

The mitogen-activated protein kinases (MAPKs) play a central role in mediating the activation and transcriptional responses of diverse cells, including glia. c-Jun N-terminal kinase (JNK), a member of the MAPK family, is activated by a variety of stress and proinflammatory signals and in turn phosphorylates its downstream substrates including nuclear factors, leading to transcriptional activation of target genes. There are at least three subtypes of JNK (i.e., JNKs 1-3) that may play isoform-specific roles. This study examined the role of JNK isoforms in the induction of inducible nitric oxide synthase (iNOS) in astrocytes in response to lipopolysachharide (LPS) and interferon (IFN)-gamma. While an inhibitor of the JNK pathway (SP600125) inhibited iNOS expression, ectopic expression of a constitutively active form of MEKK1 (MAPK/ERK kinase kinase- 1), an upstream activator of JNK, led to an induction of co-transfected iNOS promoter activity and, in the presence of LPS, to an enhanced expression of iNOS. RNA knockdown studies with JNK subtype-specific short-interfering RNA (siRNA), indicated that JNK1- but not JNK2- nor JNK3-specific siRNA, interfered with LPS/IFNgamma induction of iNOS. It is concluded that, of the three JNK forms, JNK1 is the major mediator of iNOS induction and perhaps, inflammatory signaling in general, in glial cells.

Protein Kinase D Specifically Mediates Apoptosis Signal-regulating Kinase 1-JNK Signaling Induced by H2O2 but Not Tumor Necrosis Factor

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.

JNK Regulates the Release of Proapoptotic Mitochondrial Factors in Reovirus-Infected Cells

Reovirus-induced apoptosis is associated with activation of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) and the JNK-associated transcription factor c-Jun. Here we show that reovirus-induced apoptosis and activation of caspase 3 are inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells. Inhibition of JNK activity following reovirus infection delays the release of proapoptotic mitochondrial factors and the subsequent onset of apoptosis. In contrast, reovirus-induced apoptosis is not blocked by infection with adenovirus expressing dominant-negative c-Jun, and c-Jun activation does not correlate with apoptosis in reovirus-infected cells. This is the first report demonstrating that JNK is associated with regulation of mitochondrial pathways of apoptosis following viral infection.

Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway.

Sodium 4-phenylbutyrate (PB) has been used in the therapy of urea cycle defects for many years. Recently, it has been shown to cause cellular differentiation, growth arrest, and apoptosis in certain malignancies. We have analyzed the effects of PB on human lung carcinoma cells. PB has distinct patterns of effects on different lung carcinoma cells, inducing apoptosis in NCI-H460 and NCI-H1792 cells, causing G1 arrest in A549 and SK-LU-1 cells, but having no effect on a non-transformed bronchial epithelial cell line HBE4-E6/E7. We investigated the role of MAP kinase family members, extracellular signal-regulated kinase (ERK), JNK, and p38 mitogen-activated protein kinase (MAPK), as well as other important cell survival signaling molecules in PB-induced apoptosis. We observed activation of JNK and ERK by PB in the lung cancer cells. JNK was activated only in the two apoptotic cells, whereas ERK was activated in both the apoptotic and the growth-arrested cells, demonstrating a correlation between apoptosis and activation of JNK in response to PB. Both JNK inhibitor and JNK RNA interference (RNAi) inhibited PB-induced apoptosis, whereas MEK inhibitor did not, supporting that apoptosis induced by PB is through activation of JNK. De novo protein synthesis is required for the PB-induced JNK activation and induction of apoptosis. However, the production of known upstream activators of JNK, namely Fas/Fas ligand, tumor necrosis factor (TNF)-alpha, TNF-beta, and TRAIL, are not altered by PB treatment. Therefore, PB activates JNK through an unidentified and cell type-specific mechanism. Understanding of this mechanism is of therapeutic value in treating cancer patients with PB.

Roles of TRP14, a Thioredoxin-related Protein in Tumor Necrosis Factor-α Signaling Pathways

The possible roles of a 14-kDa human thioredoxin (Trx)-related protein (TRP14) in TNF-alpha signaling were studied in comparison with those of Trx1 by RNA interference in HeLa cells. Depletion of TRP14 augmented the TNF-alpha-induced phosphorylation and degradation of I kappa B alpha as well as the consequent activation of NF-kappa B to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-alpha-induced activation of caspases and subsequent apoptosis by a similar extent. The TNF-alpha-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of apoptosis signal-regulating kinase-1 (ASK1), an upstream activator of JNK and p38. In combination with the results in the accompanying paper that TRP14 did not reduce the known substrates of Trx1, these results suggest that TRP14 modulates TNF-alpha signaling pathways, provably by interacting with proteins distinct from the targets of Trx1. In an effort to identify target proteins of TRP14, a mutant of TRP14, in which the active site cysteine (Cys(46)) was substituted with serine, was shown to form a disulfide-linked complex with LC8 cytoplasmic dynein light chain. The complex was detected in HeLa cells treated with H(2)O(2) or TNF-alpha but not in untreated cells, suggesting that LC8 cytoplasmic dynein light chain is a possible substrate of TRP14.

Evidence That Phosphatidylinositol 3-Kinase- and Mitogen-activated Protein Kinase Kinase-4/c-Jun NH2-terminal Kinase-dependent Pathways Cooperate to Maintain Lung Cancer Cell Survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.

Expression of Angiogenic Factor Cyr61 during Neuronal Cell Death via the Activation of c-Jun N-terminal Kinase and Serum Response Factor

The immediate early gene, cyr61, is transcriptionally activated within minutes by serum and serum growth factors. The encoded Cyr61 protein is secreted into the extracellular matrix and promotes cell adhesion and migration. In this study, we sought to examine the expression profile of cyr61 gene during neuronal cell death induced by various toxic stimuli and the mechanisms involved. Our data show that toxic stimuli, such as etoposide, significantly increased cyr61 mRNA levels in immortalized hippocampal progenitor (H19-7) cells. Cyr61 transcriptional activation was corroborated at the protein level as well. To identify the upstream signaling cascades involved in cyr61 gene induction, the blocking effect of either JNK or p38 kinase-signaling pathway on cyr61 induction in response to etoposide was tested. Transfection of the cells with a kinase-deficient mutant MEKK, an upstream activator of JNK, significantly decreased the cyr61 expression induced by etoposide. In contrast, cyr61 mRNA levels did not change after pretreatment with SB203580, the p38 kinase inhibitor. When the induction of cyr61 was tested by using several of its deleted promoters driving the expression of reporter gene, the promoter activation occurred primarily within the region containing an SRE-like CArG box. In addition, the SRF, which binds to the CArG site, was directly phosphorylated by active JNK. Furthermore, the blockade of cyr61 gene expression using an antisense encoding cyr61 sequence significantly inhibited the cell death induced by etoposide. Overall, these results suggest that the induction of the immediate early gene, cyr61, is important for neuronal cell death in the central nervous system hippocampal progenitor cells, and JNK activation, but not of p38, as well as the subsequent SRF phosphorylation are involved in cyr61 gene induction.

Mixed lineage kinase 3 mediates gp120IIIB-induced neurotoxicity.

Overexpression of gp120, the major coat protein of the HIV-1 virus, in central glial cells, or treatment of neurons with gp120 in culture, produces apoptotic neuronal death. Here we demonstrate that CEP-1347 (KT7515), an inhibitor of mixed lineage kinase 3 (MLK3), an upstream activator of JNK, inhibits gp120IIIB-induced apoptosis of hippocampal neurons. Furthermore, expression of wild type MLK3 in hippocampal pyramidal neurons enhanced gp120IIIB-induced neurotoxicity, whereas expression of a dominant negative MLK3 protected neurons from the toxic effects of the glycoprotein. These results indicate a role for MLK3 signaling in gp120IIIB-induced neuronal death, and suggest potential clinical utility of CEP-1347 in inhibiting the progression of AIDS dementia.

A Novel Specific Role for IκB Kinase Complex-associated Protein in Cytosolic Stress Signaling

We demonstrate here a novel role for the I kappa B kinase complex-associated protein (IKAP) in the regulation of activation of the mammalian stress response via the c-Jun N-terminal kinase (JNK)-signaling pathway. We cloned IKAP as a JNK-associating protein using the Ras recruitment yeast two-hybrid system. IKAP efficiently and specifically enhanced JNK activation induced by ectopic expression of MEKK1 and ASK1, upstream activators of JNK. Importantly, IKAP also enhanced JNK activation induced by ultraviolet light irradiation as well as treatments with tumor necrosis factor or epidermal growth factor. The JNK association site in IKAP was mapped to the C-terminal part of IKAP. Interestingly, this region is deleted from IKAP expressed in the autonomous nervous system of the patients affected by familial dysautonomia. Ectopic expression of this C-terminal fragment of IKAP was sufficient to support JNK activation. Taken together, our data demonstrate a novel role for IKAP in the regulation of the JNK-mediated stress signaling. Additionally, our results point to a role of JNK signaling in familial dysautonomia and, thus, further support the involvement of JNK signaling in the development, survival, and degeneration of the sensory and autonomic nervous system.