JNK Activation In Response Research Articles

Stress pathway outputs are encoded by pH-dependent clustering of kinase components

Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

Obesity is a major risk factor for the development ofmetabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH2-terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV andthe subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

Targeting F2r/Beta-Catenin/JNK Signaling for Leukemia Stem Cell Eradication

G-protein coupled receptors (GPCRs) are the most successful drug targets with 36% of currently marketed drugs targeting human GPCRs. F2r, a GPCR that is overexpressed in various human cancers (Ribeiro et al., Oncol Rep 2009), is a positive regulator of the β-catenin pathway and an inhibitor of the JNK pathway in mammalian cells (Sun et al., Nat Cell Biol 2001). The expression of F2r is significantly elevated in aggressive leukemias including blast phase of CML and AML (Veiga et al., Blood Cells Mol Dis 2011). While these observations implicate an involvement of F2r in human leukemia, its function in AML remains unknown.Our preliminary data showed that shRNA-mediated knockdown of F2r markedly decreased active β-catenin in MLL-AF9 mediated pre-leukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs), confirming that F2r is a positive regulator of β-catenin. F2r deficiency decreased LSC colony formation (p=0.0002) in a serial replating assay, indicating a reduction in self-renewal capacity. Furthermore, LSCs exhibited a significant enhancement in apoptotic activity in response to F2r deficiency, displaying a 12-fold increase in apoptosis. Our microarray expression analysis revealed that F2r inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). To investigate this further we used a mitochondria-specific fluorogenic probe to measure reactive oxygen species (ROS) production. A significant increase in ROS production (p=0.0003) indicated that F2r inhibition destabilizes the mitochondrial membrane. This was accompanied by a marked increase, as observed by Western blot analysis, in the proapoptotic proteins Bcl-2-interacting mediator of cell death (Bim) and thioredoxin-interacting protein (Txnip) which permeabilize the mitochondrial membrane releasing cytochrome c and inducing apoptosis. Through oxidative phosphorylation, mitochondria play an essential role in the supply of metabolic energy (ATP) to the cell. F2r deficient LSCs had a significantly reduced rate of oxygen consumption measured using a phosphorescent oxygen probe (p=0.0066) and a significantly lower concentration of basal ATP (p=0.012) compared to control LSCs. F2r inhibition, therefore, induces substantial oxidative stress which triggers the intrinsic apoptotic pathway.To assess the therapeutic value of F2r inhibition, we used the selective non-peptide F2r inhibitor SCH79797. Alamar blue-based cell viability assays showed that SCH79797 was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells. F2r inhibitor treatment resulted in a 2-fold reduction in colony forming ability, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labeling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with SCH79797 for 48 hours in culture, transplanted into recipient mice and collected from bone marrow 8 days post-transplantation (p=0.0008). Additional in vivo studies using a mouse model of MLL AML are currently ongoing to further evaluate the therapeutic potential of F2r inhibition. Collectively, our findings suggest that F2r inhibition selectively targets LSC self-renewal, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells.Previous studies suggest that F2r knockdown not only suppresses β-catenin but also activates JNK signaling. Consistently, our Western blot analysis revealed activation of JNK in response to inhibition of F2r. Sustained JNK activation has been reported in many types of AML cells and promotes survival signals during leukemia development (Hess et al., Nat Genet 2002). This suggests that JNK and F2r inhibition could be used in combination to impair LSC self-renewal, with a concurrent increase in cell death. In support of this hypothesis, we have showed that co-inhibition of F2r and JNK induced a potent anti-LSC effect, significantly increasing cell death and ROS production compared to single treatment. The efficacy of this co-treatment is currently being evaluated in primary human AML patient samples in addition to our in vivo mouse model system. Altogether our data suggest a novel LSC-eliminating treatment strategy targeting F2r/β-catenin/JNK signaling for aggressive AML. DisclosuresNo relevant conflicts of interest to declare.

Overexpressed DNA Polymerase Iota Regulated by JNK/c-Jun Contributes to Hypermutagenesis in Bladder Cancer

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis.

MLK3 promotes metabolic dysfunction induced by saturated fatty acid-enriched diet

Saturated fatty acids activate the c-Jun NH₂-terminal kinase (JNK) pathway, resulting in chronic low-grade inflammation and the development of insulin resistance. Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in response to saturated fatty acids in vitro; however, the exact mechanism for diet-induced JNK activation in vivo is not known. Here, we have used MLK3-deficient mice to examine the role of MLK3 in a saturated-fat diet model of obesity. MLK3-KO mice fed a high-fat diet enriched in medium-chain saturated fatty acids for 16 wk had decreased body fat compared with wild-type (WT) mice due to increased energy expenditure independently of food consumption and physical activity. Moreover, MLK3 deficiency attenuated palmitate-induced JNK activation and M1 polarization in bone marrow-derived macrophages in vitro, and obesity induced JNK activation, macrophage infiltration into adipose tissue, and expression of proinflammatory cytokines in vivo. In addition, loss of MLK3 improved insulin resistance and decreased hepatic steatosis. Together, these data demonstrate that MLK3 promotes saturated fatty acid-induced JNK activation in vivo and diet-induced metabolic dysfunction.

Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: evidence for non-canonical activation of RhoA and Rac1 GTPases

Although statins are known to inhibit proliferation and induce death in a number of cancer cell types, the mechanisms through which downregulation of the mevalonate (MVA) pathway activates death signaling remain poorly understood. Here we set out to unravel the signaling networks downstream of the MVA pathway that mediate the death-inducing activity of simvastatin. Consistent with previous reports, exogenously added geranylgeranylpyrophosphate, but not farnesylpyrophosphate, prevented simvastatin's growth-inhibitory effect, thereby suggesting the involvement of geranylgeranylated proteins such as Rho GTPases in the anticancer activity of simvastatin. Indeed, simvastatin treatment led to increased levels of unprenylated Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42). Intriguingly, instead of inhibiting the functions of Rho GTPases as was expected with loss of prenylation, simvastatin caused a paradoxical increase in the GTP-bound forms of RhoA, Rac1 and Cdc42. Furthermore, simvastatin disrupted the binding of Rho GTPases with the cytosolic inhibitor Rho GDIα, which provides a potential mechanism for GTP loading of the cytosolic Rho GTPases. We also show that the unprenylated RhoA- and Rac1-GTP retained at least part of their functional activities, as evidenced by the increase in intracellular superoxide production and JNK activation in response to simvastatin. Notably, blocking superoxide production attenuated JNK activation as well as cell death induced by simvastatin. Finally, we provide evidence for the involvement of the B-cell lymphoma protein 2 family, Bcl-2-interacting mediator (Bim), in a JNK-dependent manner, in the apoptosis-inducing activity of simvastatin. Taken together, our data highlight the critical role of non-canonical regulation of Rho GTPases and involvement of downstream superoxide-mediated activation of JNK pathway in the anticancer activity of simvastatin, which would have potential clinical implications.

PI3K/Akt-independent negative regulation of JNK signaling by MKP-7 after cerebral ischemia in rat hippocampus

BackgroundThe inactivation of c-Jun N-terminal kinase (JNK) is associated with anti-apoptotic and anti-inflammatory effects in cerebral ischemia, which can be induced by an imbalance between upstream phosphatases and kinases.ResultMitogen-activated protein kinase phosphatase 7 (MKP-7) was upregulated significantly at 4 h of reperfusion postischemia in rat hippocampi. By administration of cycloheximide or siRNA against mitogen-activated protein kinase phosphatase 7 (MKP-7) in a rat model of ischemia/reperfusion, an obvious enhancement of JNK activity was observed in 4 h of reperfusion following ischemia, suggesting MKP-7 was involved in JNK inactivation after ischemia. The subcellular localization of MKP-7 altered after ischemia, and the inhibition of MKP-7 nuclear export by Leptomycin B up-regulated JNK activity. Although PI3K/Akt inhibition could block downregulation of JNK activity through SEK1 and MKK-7 activation, PI3K/Akt activity was not associated with the regulation of JNK by MKP-7.ConclusionsMKP-7, independently of PI3K/Akt pathway, played a key role in downregulation of JNK activity after ischemia in the rat hippocampus, and the export of MKP-7 from the nucleus was involved in downregulation of cytoplasmic JNK activity in response to ischemic stimuli.

Critical Role for Mixed-Lineage Kinase 3 in Acetaminophen-Induced Hepatotoxicity

c-Jun NH(2)-terminal kinase (JNK) activation plays a major role in acetaminophen (APAP)-induced hepatotoxicity. However, the exact mechanism of APAP-induced JNK activation is incompletely understood. It has been established that apoptosis signal-regulating kinase 1 (ASK1) regulates the late phase of APAP-induced JNK activation, but the mitogen-activated protein kinase kinase kinase that mediates the initial phase of APAP-induced JNK activation has not been identified. Oxidative stress produced during APAP metabolism causes JNK activation, which promotes mitochondrial dysfunction and results in the amplification of oxidative stress. Therefore, inhibition of the initial phase of JNK activation may be key to protection against APAP-induced liver injury. The goal of this study was to determine whether mixed-lineage kinase 3 (MLK3) mediates the initial, ASK1-independent phase of APAP-induced JNK activation and thus promotes drug-induced hepatotoxicity. We found that MLK3 was activated by oxidative stress and was required for JNK activation in response to oxidative stress. Loss of MLK3 attenuated APAP-induced JNK activation and hepatocyte death in vitro, independent of receptor-interacting protein 1. Moreover, JNK and glycogen synthase kinase 3β activation was significantly attenuated, and Mcl-1 degradation was inhibited in APAP-treated MLK3-knockout mice. Furthermore, we showed that loss of MLK3 increased expression of glutamate cysteine ligase, accelerated hepatic GSH recovery, and decreased production of reactive oxygen species after APAP treatment. MLK3-deficient mice were significantly protected from APAP-induced liver injury, compared with wild-type mice. Together, these studies establish a novel role for MLK3 in APAP-induced JNK activation and hepatotoxicity, and they suggest MLK3 as a possible target in the treatment of APAP-induced liver injury.

Aβ Potentiates Inflammatory Activation of Glial Cells Induced by Scavenger Receptor Ligands and Inflammatory Mediators in Culture

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the accumulation of β amyloid (Aβ) aggregates. Aβ induces the inflammatory activation of glia, inducing secretion of Interleukin 1β (IL1β), nitric oxide (NO) and superoxide radicals. The specific receptor responsible for the induction of inflammatory activation by Aβ, is still an open question. We propose that scavenger receptors (SR) participate in the activation of glia by Aβ. We assessed production of NO, synthesis of IL1β and activation of ERK, JNK and NF-κB signaling pathways by Western blot, in primary rat glial cultures exposed to SR ligands (fucoidan and Poly I), LPS+IFNγ (LI), and Aβ. Poly I but not fucoidan nor fibrillar Aβ increased threefold NO production by astrocytes in a time-dependent manner. Fucoidan and Poly I increased 5.5- and 3.5-fold NO production by microglia, and co-stimulation with Aβ increased an additional 60% NO induced by SR ligands. Potentiation by Aβ was observed later for astrocytes than for microglia. In astrocytes, co-stimulation with Aβ potentiated ERK and JNK activation in response to Fucoidan and Poly I, whereas it reduced induction of JNK activation by LI and left unaffected NF-κB activation induced by LI. Levels of pro-IL1β in astrocytes increased with Aβ, SR ligands and LI, and were potentiated by co-stimulation with Aβ. Our results suggest that SRs play a role on inflammatory activation, inducing production of NO and IL1β, and show potentiation by Aβ. Potentiation of the inflammatory response of Aβ could be meaningful for the activation of glia observed in AD.

Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses

C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.

Phosphorylation of the M3/6 dual-specificity phosphatase enhances the activation of JNK by arsenite

Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway critically depend on the intensity and duration of signal transmission. Dual-specificity phosphatases (DUSPs) play a very important role in these events by modulating the extent of JNK phosphorylation and activation and thus regulating cellular responses to stress. M3/6 (DUSP8) is one of the dual-specificity protein phosphatases with distinct specificity towards JNK. It has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with arsenite, but the role of this phosphorylation has not been investigated. In this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were identified and site-directed mutagenesis was employed to investigate their role. Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased phosphorylation compared to the wild-type protein. No difference was observed in terms of the enzyme's in vitro phosphatase activity, its substrate specificity towards JNK isoforms, its interactions with JNK and the scaffold family of JNK-interacting proteins (JIPs), its stability or its subcellular localization. Interestingly, expression of M3/6 phosphorylation mutants delayed the time-course of JNK phosphorylation and activation by arsenite. We propose that phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli results in attenuation of phosphatase activity and acceleration of JNK activation.

Abstract 198: TRAF2 phosphorylation is essential for cancer-cell adaptation to chronic cellular stress

Abstract The tumor microenvironment is known to trigger chronic endoplasmic reticulum (ER) and oxidative stresses, and cancer-cell adaptation to such chronic stresses has profound consequences for malignant progression and the response to therapy. TRAF2 regulates cellular response to TNFα, ER and oxidative stresses by activating the JNK/c-Jun and IKK/NF-κB signaling cascades. We mapped two phosphorylation sites (Ser-11 and Ser-55) on the N-terminal RING domain of TRAF2, and reported that phosphorylation at these sites increases basal and TNFα-induced NF-κB activation. Recently, we identified TBK1 and PKCζ as the kinases that directly phosphorylate TRAF2 at Ser-11 and Ser-55 sites, respectively. We found that in addition to TNFα, ER and oxidative stresses also strongly induce TRAF2 phosphorylation at both sites. This dual phosphorylation of TRAF2 promoted IKK activation and inhibited the prolonged phase of JNK activation in response to ER and oxidative stresses. Importantly, TRAF2 phosphorylation was found to be essential for cancer-cell survival under conditions of chronic cellular stress. Specifically, the endogenous TRAF2-dockdown cancer cells that were stable expressed with phospho-null-mutant TRAF2 (TRAF2-S11/55A) did not survive under conditions of chronic ER and oxidative stresses, while their TRAF2-WT counterparts survived under the same conditions. In addition, the TRAF2-S11/55A-expressing cancer cells formed significantly smaller tumors in vivo and exhibited greater sensitivity to anti-cancer drug-induced apoptosis than did TRAF2-WT-expressing counterparts. Functional assays revealed that TRAF2 phosphorylation not only promotes NF-κB-dependent gene expression, but also suppresses JNK-mediated degradation of Bcl-2 and cIAP1 at post-transcriptional level in the context of ER and oxidative stresses. Collectively, these findings suggest that TRAF2 phosphorylation plays a critical role in tumor progression by promoting the expression of anti-apoptotic proteins in cancer cells, and could be a new target for anti-cancer drug development. 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 198. doi:10.1158/1538-7445.AM2011-198

Regulation of JNK activity in the apoptotic response of intestinal epithelial cells

We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

A kinematic model coupling stress fiber dynamics with JNK activation in response to matrix stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c-jun N-terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre-extended to a preferred level under static conditions and that perturbations from this preferred level destabilize the stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK activation as a result of the formation of new integrin bonds within the associated focal adhesions. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. In the case of cyclic uniaxial stretching, stretch-induced JNK activation slowly subsides as stress fibers gradually reorient perpendicular to the stretch direction. In contrast, JNK activation is chronically elevated in response to cyclic equibiaxial stretch. A step change in either uniaxial or equibiaxial stretch results in a short, transient upregulation in JNK that quickly returns to the basal level as overly stretched stress fibers disassemble and are replaced by fibers assembled at the preferred level of stretch. In summary, the model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces through turnover and reorganization to modulate intracellular signaling.

Sustained c-Jun-NH2-Kinase Activity Promotes Epithelial-Mesenchymal Transition, Invasion, and Survival of Breast Cancer Cells by Regulating Extracellular Signal-Regulated Kinase Activation

The c-Jun NH(2)-terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2-mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs.

Novel Role for Aldose Reductase in Mediating Acute Inflammatory Responses in the Lung

Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-alpha-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-alpha-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation.

The Logic of EGFR/ErbB Signaling: Theoretical Properties and Analysis of High-Throughput Data

The epidermal growth factor receptor (EGFR) signaling pathway is probably the best-studied receptor system in mammalian cells, and it also has become a popular example for employing mathematical modeling to cellular signaling networks. Dynamic models have the highest explanatory and predictive potential; however, the lack of kinetic information restricts current models of EGFR signaling to smaller sub-networks. This work aims to provide a large-scale qualitative model that comprises the main and also the side routes of EGFR/ErbB signaling and that still enables one to derive important functional properties and predictions. Using a recently introduced logical modeling framework, we first examined general topological properties and the qualitative stimulus-response behavior of the network. With species equivalence classes, we introduce a new technique for logical networks that reveals sets of nodes strongly coupled in their behavior. We also analyzed a model variant which explicitly accounts for uncertainties regarding the logical combination of signals in the model. The predictive power of this model is still high, indicating highly redundant sub-structures in the network. Finally, one key advance of this work is the introduction of new techniques for assessing high-throughput data with logical models (and their underlying interaction graph). By employing these techniques for phospho-proteomic data from primary hepatocytes and the HepG2 cell line, we demonstrate that our approach enables one to uncover inconsistencies between experimental results and our current qualitative knowledge and to generate new hypotheses and conclusions. Our results strongly suggest that the Rac/Cdc42 induced p38 and JNK cascades are independent of PI3K in both primary hepatocytes and HepG2. Furthermore, we detected that the activation of JNK in response to neuregulin follows a PI3K-dependent signaling pathway.

A Kinematic Model Coupling Cytoskeletal Dynamics with JNK Activation in Response to Matrix Stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c‐jun N‐terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre‐extended at a "homeostatic" level under normal, non‐perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK phosphorylation as a result of the formation of new integrin bonds within the associated focal adhesions, while the rate of JNK dephosphorylation is independent of the stretch signal. Disassembly of overly stretched stress fibers and reassembly of stress fibers at the "homeostatic" level lead to a decrease in the rates of integrin turnover and JNK activation. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. This model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces to modulate intracellular signaling.

10-Formyltetrahydrofolate Dehydrogenase–Induced c-Jun-NH2-Kinase Pathways Diverge at the c-Jun-NH2-Kinase Substrate Level in Cells with Different p53 Status

10-Formyltetrahydrofolate dehydrogenase (FDH) suppresses cancer cell proliferation through p53-dependent apoptosis but also induces strong cytotoxicity in p53-deficient prostate cells. In the present study, we have shown that FDH induces apoptosis in PC-3 prostate cells through simultaneous activation of the c-Jun-NH(2)-kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways with JNK phosphorylating c-Jun and ERK1/2 phosphorylating Elk-1. The JNK1/2 inhibitor SP600125 or ERK1/2 inhibitor PD98059 prevented phosphorylation of c-Jun and Elk-1, correspondingly and partially protected PC-3 cells from FDH-induced cytotoxicity. Combination of the two inhibitors produced an additive effect. The contribution from the JNK cascade to FDH-induced apoptosis was significantly stronger than from the ERK pathway. siRNA knockdown of JNK1/2 or "turning off" the downstream target c-Jun by either siRNA or expression of the dominant-negative c-Jun mutant, TAM67, rescued PC-3 cells from FDH-induced apoptosis. The pull-down assays on immobilized c-Jun showed that c-Jun is directly phosphorylated by JNK2 in FDH-expressing cells. Interestingly, the FDH-induced apoptosis in p53-proficient A549 cells also proceeds through activation of JNK1/2, but the down-stream target for JNK2 is p53 instead of c-Jun. Furthermore, in A549 cells, FDH activates caspase 9, whereas in PC-3 cells, it activates caspase 8. Our studies indicate that the JNK pathways are common downstream mechanisms of FDH-induced cytotoxicity in different cell types, whereas the end point target in the cascade is cell type specific. JNK activation in response to FDH was inhibited by high supplementation of reduced folate leucovorin, further indicating a functional connection between folate metabolism and mitogen-activated protein kinase pathways.