Increased Phosphorylation Of JNK Research Articles

Pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the IGF–IGFBP axis

The IGF axis is critical for the regulation of apoptosis in many human cancer cell lines. Recently, potent anti-tumorigenic effects of pomegranate juice and extracts have been reported. Consequently, pomegranate has potential not only as a treatment but also as a preventative measure against certain types of cancer, including prostate. In this study, we investigated the relationship between pomegranate-induced apoptosis in human prostate cancer cells and the IGF/IGFBP system. Treatment of LAPC4 prostate cancer cells with 10 μg/ml POMx, a highly potent pomegranate extract prepared from skin and arils minus seeds and standardized to ellagitannin content (37% punicalagins by HPLC), resulted in inhibition of cell proliferation and induction of apoptosis. Interestingly, co-treatment with POMx and IGFBP-3 revealed synergistic stimulation of apoptosis and additive inhibition of cell growth. Western blot analysis revealed that treatment with POMx or POMx/IGFBP-3 combination resulted in increased JNK phosphorylation, and decreased Akt and mTOR activation, consistent with a growth inhibitory, pro-apoptotic function. We also investigated the relationship between IGF-1 and pomegranate-induced apoptosis in 22RV1 prostate cancer cells. Co-treatment with 100 ng/ml IGF-1 completely blocked apoptosis induction by POMx. In contrast, IGF-I failed to inhibit POMx-induced apoptosis in R − cells, suggesting the importance of IGF-IR. POMx-treatment decreased Igf1 mRNA expression in a dose-dependent manner indicating that its actions also involve tumor-specific suppression of IGF-1. These studies revealed novel interactions between the IGF system and pomegranate-induced apoptosis.

Thrombin-Induced Connective Tissue Growth Factor Expression in Human Lung Fibroblasts Requires the ASK1/JNK/AP-1 Pathway

Thrombin plays an important role in lung inflammatory diseases. Thrombin can induce connective tissue growth factor (CTGF) expression in lung fibroblasts. However, little is known about the signaling pathway in thrombin-induced CTGF expression. In this study, we investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in thrombin-induced CTGF expression in human lung fibroblasts. Thrombin caused a concentration- and time-dependent increase in CTGF expression in WI-38 cells and primary lung fibroblasts. Thrombin-induced CTGF expression and CTGF-luciferase activity were inhibited by a protease-activated receptor 1 antagonist (SCH79797), the dominant-negative mutants (DNs) of ASK1 and JNK1/2, and an AP-1 inhibitor (curcumin). Thrombin caused ASK1 Ser(967) dephosphorylation, the dissociation of ASK1 and 14-3-3, and a subsequent increase in ASK1 activity. Thrombin induced increases in JNK phosphorylation and kinase activity, which were attenuated by ASK1DN. Furthermore, SCH79797 diminished the thrombin-induced ASK1 and JNK activities. Thrombin-induced CTGF-luciferase activity was predominately controlled by the sequence -747 to -184 bp upstream of the transcription start site of the human CTGF promoter and was attenuated by transfection with the deleted AP-1 binding site construct. Thrombin caused increases in c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and the recruitment of c-Jun to the CTGF promoter. Furthermore, thrombin-mediated AP-1 activation was inhibited by ASK1DN, JNK1/2DN, and SP600125. These results suggest for the first time that thrombin, acting through protease-activated receptor 1, activates the ASK1/JNK signaling pathway, which in turn initiates c-Jun/AP-1 activation and recruitment of c-Jun to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts.

Dynasore, a Dynamin Inhibitor, Induces PAI-1 Expression in MeT-5A Human Pleural Mesothelial Cells

Plasminogen activator inhibitor-1 (PAI-1) is a primary regulator of plasminogen activation that plays an essential role in regulating the physiological thrombotic/fibrinogenic balance. The elevation of PAI-1 expression by human pleural mesothelial cells has been reported to contribute to pleural fibrosis and pleurodesis. In this study, we examined the effects on PAI-1 expression of dynasore, a cell-permeable inhibitor of dynamin, and its mechanisms in a human pleural mesothelial cell line (MeT-5A). The results indicated that dynasore enhanced transforming growth factor (TGF)-beta(1)- and TNF-alpha-induced PAI-1 protein expression in a concentration-dependent manner. Furthermore, dynasore significantly up-regulated PAI-1 protein and its messenger RNA expressions. Interestingly, Smad2/3 activation was induced by TGF-beta(1) but not by dynasore. Among signaling inhibitors, a c-Jun NH(2)-terminal kinase (JNK) inhibitor (SP600125) markedly attenuated dynasore-stimulated PAI-1 protein production. Consistently, dynasore strongly increased JNK phosphorylation. On the other hand, there was no enhancement effect by dynasore on TGF-beta(1)-induced matrix metalloproteinase-2 activation. These findings suggest that dynasore may stimulate PAI-1 protein expression and enhance TGF-beta(1) activity through activation of JNK-mediated signaling in human pleural mesothelial cells. Given the profibrotic effect of dynasore, further in vivo studies may be conducted to evaluate its potential as a pleurodesing agent.

Proteomics and Pathway Analysis Identifies JNK Signaling as Critical for High Linear Energy Transfer Radiation-induced Apoptosis in Non-small Lung Cancer Cells

During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p = 6.10(-6)) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p = 3.10(-5)) and the p38 pathway was found to be deactivated (p = 0.001) compared with untreated cells. Antibody-based analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool.

498 Autocrine Intestinal Epithelial Cell GM-CSF Signaling Promotes Homeostatic Responses to Gut Injury

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship

497 ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship

Blockade of the RhoA-JNK-c-Jun-MMP2 Cascade by Atorvastatin Reduces Osteosarcoma Cell Invasion

Osteosarcoma is characterized by a high malignant and metastatic potential, which points to the need for new therapeutic strategies to prevent cell metastasis. In this study, we show that statin-induced HMG-CoA reductase inhibition reduces cell migration and invasion in human and murine osteosarcoma cells, independently of the genotype. The statin-induced reduction of cell migration and invasion was independent of induction of apoptosis and was geranylgeranylpyrophosphate-dependent. The statin reduced matrix metalloproteinase (MMP) 2, 9, and 14 and TIMP2 expression or activity in invading cells. Forced expression of MMP2 and MMP14 overcame the inhibitory effect of the statin on cell invasion, suggesting a role for these MMPs in invasive potential. We also investigated the mechanisms involved in the reduced MMP2 activity and cell invasion. Inhibition of JNK, but not ERK1/2 signaling, reduced MMP2 activity. Pharmacological or constitutive activation of JNK overcame the reduced MMP2 activity and cell invasion induced by the statin. The statin decreased JNK phosphorylation and c-Jun nuclear translocation, suggesting that HMG-CoA reductase inhibition targets the JNK-c-Jun signaling pathway. We showed that mevalonate or geranylgeranylpyrophosphate treatment prevented the statin-induced reduction in JNK phosphorylation, MMP2 activity, and cell invasion. Forced expression of a constitutively active form of RhoA increased JNK phosphorylation and overcame the inhibitory effect of atorvastatin on MMP2 activity and cell invasion. The data establish a link between RhoA, JNK, c-Jun, and MMP2 activity that is functionally involved in the reduction in osteosarcoma cell invasion by the statin. This suggests a novel strategy targeting RhoA-JNK-c-Jun signaling to reduce osteosarcoma cell tumorigenesis.

C-Jun N-Terminal Kinase 2 Deficiency Protects Against Hypercholesterolemia-Induced Endothelial Dysfunction and Oxidative Stress

Hypercholesterolemia-induced endothelial dysfunction due to excessive production of reactive oxygen species is a major trigger of atherogenesis. The c-Jun-N-terminal kinases (JNKs) are activated by oxidative stress and play a key role in atherogenesis and inflammation. We investigated whether JNK2 deletion protects from hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Male JNK2 knockout (JNK2(-/-)) and wild-type (WT) mice (8 weeks old) were fed either a high-cholesterol diet (HCD; 1.25% total cholesterol) or a normal diet for 14 weeks. Aortic lysates of WT mice fed a HCD showed an increase in JNK phosphorylation compared with WT mice fed a normal diet (P<0.05). Endothelium-dependent relaxations to acetylcholine were impaired in WT HCD mice (P<0.05 versus WT normal diet). In contrast, JNK2(-/-) HCD mice did not exhibit endothelial dysfunction (96+/-5% maximal relaxation in response to acetylcholine; P<0.05 versus WT HCD). Endothelium-independent relaxations were identical in all groups. A hypercholesterolemia-induced decrease in nitric oxide (NO) release of endothelial cells was found in WT but not in JNK2(-/-) mice. In parallel, endothelial NO synthase expression was upregulated only in JNK2(-/-) HCD animals, whereas the expression of antioxidant defense systems such as extracellular superoxide dismutase and manganese superoxide dismutase was decreased in WT but not in JNK2(-/-) HCD mice. In contrast to JNK2(-/-) mice, WT HCD displayed an increase in O(2)(-) and ONOO(-) concentrations as well as nitrotyrosine staining and peroxidation. JNK2 plays a critical role as a mediator of hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Thus, JNK2 may provide a novel target for prevention of vascular disease and atherosclerosis.

Role of endoplasmic reticulum stress in acrolein-induced endothelial activation

Acrolein is a ubiquitous environmental pollutant and an endogenous product of lipid peroxidation. It is also generated during the metabolism of several drugs and amino acids. In this study, we examined the effects of acrolein on endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with 2 to 10 μM acrolein led to an increase in the phosphorylation of eIF-2α within 10 to 30 min of exposure. This was followed by alternate splicing of XBP-1 mRNA and an increase in the expression of the endoplasmic reticulum (ER) chaperone genes Grp78 and Herp. Within 2–4 h of treatment, acrolein also increased the abundance and the nuclear transport of the transcription factors ATF3, AFT4, and CHOP. Acrolein-induced increase in ATF3 was prevented by treating the cells with the chemical chaperone — phenylbutyric acid (PBA). Treatment with acrolein increased phosphorylation of ERK1/2, p38, and JNK. The increase in JNK phosphorylation was prevented by PBA. Acrolein treatment led to activation and nuclear translocation of the transcription factor NF-κB and an increase in TNF-α, IL-6 and IL-8, but not MCP-1, mRNA. Increased expression of cytokine genes and NF-κB activation were not observed in cells treated with PBA. These findings suggest that exposure to acrolein induces ER stress and triggers the unfolded protein response and that NF-κB activation and stimulation of cytokine production by acrolein could be attributed, in part, to ER stress. Chemical chaperones of protein-folding may be useful in treating toxicological and pathological states associated with excessive acrolein exposure or production.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through activation of c-jun N-terminal kinase

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77 and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para >/= meta >/= ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose) polymerase (PARP) that is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with the activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK.

Sodium Tanshinone IIA Sulfonate Protects Cardiomyocytes Against Oxidative Stress-mediated Apoptosis Through Inhibiting JNK Activation

Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a well-known Chinese medicine for treating cardiovascular disorders. Cardiomyocyte apoptosis plays a major role in the development of cardiovascular diseases. The present study was designed to investigate the effects of STS on cardiomyocyte apoptosis induced by in vivo acute myocardial infarction (MI) in adult rats and by in vitro H2O2-treated neonatal rat ventricular myocytes. In MI rats, STS significantly reduced the infarct sizes, the blood lactate dehydrogenase (LDH) level, and the number of apoptotic cardiomyocytes in the infarcted hearts. In the in vitro study, STS reversed the decreased effect of cell viability induced by H2O2. In addition, STS also markedly inhibited H2O2-induced cardiomyocyte apoptosis. C-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) and p38 MAPK are classic oxidative stress-activated protein kinases. Our further mechanistic study revealed that increased JNK phosphorylation stimulated by H2O2 was abolished by STS treatment. In conclusion, inhibition of JNK activation plays a significant role in cardioprotective effects of STS.

HSP72 protects against obesity-induced insulin resistance

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

EPO Receptor Overexpression in Breast Cancer Cells Enhances EPO Sensitivity, Induces ERK and AKT Phosphorylation and Promotes Cellular Proliferation and Migration through a JNK-Dependent Pathway.

The ubiquitous expression of the EPO receptor (EpoR) in various non-hematopoietic tissues is associated with broad EPO-mediated tissue-protective effects. The role of EpoR expression in tumor cells and its potential for contribution to tumor biology and progression requires further characterization. We investigated whether EpoR expression levels and activation in tumor cells may modulate EPO sensitivity, proliferation and cell migration. MCF-7 breast cancer cells which express endogenous EpoR were engineered to stably express R129C-EpoR, a constitutively active EpoR that confers cytokine-independent hematopoietic cell growth but does not transform fibroblasts. Empty vector-transfected cells were negative controls. In untransfected cells, phosphotyrosine immunoblots showed EPO-induced tyrosine phosphorylation of several cellular proteins. EPO resulted in dose- and time-dependent phosphorylation of ERK1/2, but not JAK2, STAT5 or AKT. Increased EPO-induced ERK phosphorylation was associated with 2.3±0.3-fold increase in ERK kinase activity (P<0.05, n=3). EPO exhibited either a minor effect or no effect on proliferation of MCF-7 cells whereas cell migration in Transwell chambers was significantly stimulated by EPO (P<0.05, n=9). R129C-EpoR expression resulted in 2-fold increase in cellular proliferation (P<0.01, 2 clones, n=6) and 1.5±0.2-fold increase in cellular migration compared to vector controls (P<0.05, 3 clones, n=12). EPO significantly stimulated the migration of empty vector-transfected cells by 1.9±0.2-fold (P<0.001, n=12). Exogenous EPO did not further enhance the increased proliferation and migration of R129C-EpoR cells. We investigated the mechanisms involved in the increased migration of R129C-EpoR cells. Basal phosphorylation of ERK1/2 (2.6±0.4-fold, 3 clones, P<0.05) and AKT (1.6±0.1-fold, P<0.05) were significantly increased compared to vector controls. No changes in the phosphorylation status of JAK2, STAT5, JAK1 or p38 MAP kinases were observed under basal conditions or in response to EPO. Whereas EPO did not induce AKT phosphorylation in untransfected or vector-transfected cells, in R129C-EpoR cells, there was a significant, dose-dependent increase in AKT phosphorylation in response to EPO (3.29±0.5-fold, n=3 clones, P<0.05). In R129C-EpoR cells, EPO significantly enhanced ERK1/2 phosphorylation 2.7±0.3 fold compared to vector controls (P<0.001). Because c-Jun-NH2-kinase (JNK) is involved in cell migration, we determined the effect of EPO on JNK phosphorylation. EPO markedly increased JNK phosphorylation in R129C-EpoR cells by 5.38±1.6 fold (P<0.001) compared to vector controls in a dose-dependent manner. JNK phosphorylation and increased EPO-induced migration of vector-transfected cells were blocked by treatment with JNK kinase inhibitor SP600125. The increased migration of R129C-EpoR cells in the absence of exogenous EPO was also blocked by SP600126. These data indicate that1.-EPO promotes the migration of breast cancer cells through a JNK-kinase dependent signaling pathway2.-Expression of R129C-EpoR promotes the proliferation and migration of breast cancer cells3.-EPO sensitivity is enhanced by R129C-EpoR expression leading to restoration of EPO-dependent AKT phosphorylation and increased ERK1/2 and JNK kinase phosphorylation4.-The biologic effects of EPO in breast cancer cells are mediated through JAK2-STAT5 axis-independent signaling pathways5.-EpoR expression levels in tumor cells may modulate tumor cell proliferation and migration responses.

Activation and Dysregulation of the Unfolded Protein Response in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) are associated with known triggers of the unfolded protein response (UPR). The aims were to (1) evaluate the activity of UPR in NAFL and NASH and (2) correlate expression of UPR pathways with liver histology. Messenger RNA (mRNA) and protein expression were measured by quantitative real-time PCR and Western blot, respectively. Apoptosis was assessed by TUNEL assay. Liver histology was scored using the NASH clinical research network criteria. Compared with subjects with the metabolic syndrome and normal liver histology (n = 17), both NAFL (n = 21) and NASH (n = 21) were associated with increased eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation. Activating transcription factor 4 (ATF4) mRNA and protein, C/EBP homologous protein (CHOP), and growth arrest, DNA damage-34 (GADD34) mRNA were not increased in NAFL or NASH. Whereas immunoglobulin heavy chain binding protein mRNA was significantly increased in NASH, unspliced X-box protein-1 (XBP-1) protein did not increase. Also, endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein mRNA levels were inversely related to spliced XBP-1 mRNA in NASH. NASH was specifically associated with low sXBP-1 protein and increased JNK phosphorylation. This correlated with increased TUNEL activity in NASH. The histologic severity correlated with sXBP-1 mRNA and JNK phosphorylation. There is a variable degree of UPR activation in NAFL and NASH. Although both NAFL and NASH are associated with eIF-2alpha phosphorylation, there is a failure to activate downstream recovery pathways, ie, ATF4-CHOP-GADD34. NASH is specifically associated with (1) failure to generate sXBP-1 protein and (2) activation of JNK.

Abstract 915: Deletion of JNK2 Prevents Hypercholesterolemia-Induced Oxidative Stress and Endothelial Dysfunction

Background Hypercholesterolemia-induced endothelial dysfunction (ED) is a major trigger for atherosclerosis. The c-Jun-N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase family. Genetic deletion of JNK2 has been shown to decrease atheroma formation. The present study was designed to investigate whether hypercholesterolemic JNK2 knockout ( JNK2 −/− ) mice are protected from oxidative stress-induced ED. Methods and Results Male JNK2 −/− and corresponding wild-type (WT) mice (8 weeks old) were fed either a high cholesterol diet (HCD, 1.25% total cholesterol) or normal chow (controls) for 14 weeks. WT mice fed a HCD showed a 2-fold increase in JNK phosphorylation as assessed by Western blotting (n=4 – 6, p&lt;0.05 vs. controls). In parallel, endothelium-dependent relaxations to acetylcholine (Ach, 10 −9 -10 −6 mol/L) were impaired in WT mice exposed to a HCD as compared to WT controls (n=4 – 6 in each groups, p&lt;0.05). In contrast, JNK2 −/− mice did not exhibit hypercholesterolemia-induced ED (96±5% max relaxation to Ach, n=4 – 6, p&lt;0.05 vs. WT HCD). Endothelium-independent relaxations to sodium nitroprusside (10 −10 -10 −5 mol/L) were identical in all groups. Nitric oxide release, measured by nanosensors in single aortic endothelial cells, was decreased in WT but not in JNK2 −/− HCD mice (121 vs. 253 nmol/L in WT and JNK2 −/− HCD mice, respectively, n=4 – 6, p&lt;0.05). Indeed, WT HCD mice displayed a marked increase in peroxynitrite (ONOO − ) generation, nitrotyrosine expression,and lipid peroxidation (n=4 – 6 in each groups, p&lt;0.05 vs. WT and JNK2 −/− control as well as JNK2 −/− HCD mice). Deletion of the JNK2 gene prevented this hypercholesterolemia-induced increase in oxidative stress. Furthermore, expression of antioxidant defense systems such as manganese and extracellular superoxide dismutase as well as heme-oxygenase activity was significantly decreased in WT, but not in JNK2 −/− HCD mice (n=4 – 6, p&lt;0.05). Conclusions These data suggest that genetic deletion of JNK2 protects against hypercholesterolemia-induced oxidative stress and endothelial dysfunction. Thus, inhibition of JNK2 may represent a novel therapeutic target to prevent initiation of atherogenesis.

C-Jun N-terminal kinase regulates adenosine A1 receptor-mediated synaptic depression in the rat hippocampus

Adenosine A1 receptors are ubiquitous mediators of presynaptic inhibition of neurotransmission in the central nervous system, yet the signalling pathway linking A1 receptor activation and decreased neurotransmitter release remains poorly resolved. We tested the contribution of c-Jun N-terminal kinase (JNK) to adenosine A1 receptor-mediated depression of field excitatory postsynaptic potentials (fEPSPs) in area CA1 of the rat hippocampus. We found that inhibition of JNK with SP600125 or JNK inhibitor V, but not an inactive analogue, attenuated the depression of fEPSPs induced by adenosine, hypoxia, and the A1 receptor agonist N 6-cyclopentyladenosine (CPA). In contrast, the JNK inhibitor SP600125 did not inhibit GABA B-mediated synaptic depression. In support of our electrophysiological findings, Western blot analysis showed that A1 receptor stimulation resulted in a transient increase in JNK phosphorylation in the membrane fraction of hippocampal lysates. The total amount of JNK in the membrane fraction was unchanged by CPA treatment. The increase in phosphorylated JNK induced by A1 receptor stimulation was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), indicating that A1 receptors specifically activate JNK in the hippocampus. Together with functional data indicating that JNK inhibition decreased CPA-induced paired pulse facilitation, these results suggest that JNK activation is necessary for adenosine A1 receptor-mediated synaptic depression occurring at a presynaptic locus The adenosine A1 receptor-JNK signalling pathway may represent a novel mechanism underlying inhibition of neurotransmitter release in the CNS.

Deletion of the c-Jun N-terminal Kinase 3 Gene Protects Neonatal Mice against Cerebral Hypoxic—Ischaemic Injury

c-Jun N-terminal kinase 3 (JNK3) is a member of the stress-activated group of mitogen-activated protein kinases. c-Jun N-terminal kinase 3 is a potent mediator of apoptosis and the use of JNK inhibitors or jnk3 gene deletion each protect against brain injury in adults. However, little is known about the role of JNK3 or its mechanism of action in neonatal brain injury. The aim of the present study was to compare the vulnerability of neonatal JNK3 knockout (JNK3 KO) mice and wild-type (WT) mice to cerebral hypoxic-ischaemic injury (HII) using unilateral-carotid occlusion combined with transient hypoxia. The degree of neural tissue loss in JNK3 KO mice was substantially reduced compared with WT mice (JNK3 KO 27.8%+/-2.8% versus WT 48.3%+/-2.0%, P<or=0.0001) after HII. Significant attenuation of injury was observed in the cerebral cortex, hippocampus, striatum, and thalamus of JNK3 KO compared with WT mice. Hypoxic-ischaemic injury increased JNK phosphorylation and activity, with JNK3 as the major isoform. Significantly, in JNK3 KO animals there was no difference in the activation of the upstream kinases mitogen-activated protein kinase kinase (MKK4) or MKK7. Downstream of JNK3, HII lead to increased phosphorylation of the transcription factors c-Jun and adenovirus transcription factor-2 (ATF-2), which was attenuated in JNK3 KO mice. c-Jun N-terminal kinase 3 deletion also decrease caspase-3 cleavage and Bim/PUMA expression, coupled with a upregulation of AKT/FOXO3a levels, linking JNK3 to apoptosis. These findings implicate JNK3 involvement in neural cell loss resulting from cerebral HII in the developing brain.

C-Jun N-terminal Kinase Contributes to Norepinephrine-Induced Contraction Through Phosphorylation of Caldesmon in Rat Aortic Smooth Muscle

Vascular smooth muscle contraction is mediated by activation of extracellular signal-regulated kinase (ERK) 1/2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dose-dependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor Gö6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by Gö6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC.

Increased JNK Phosphorylation and Oxidative Stress in Response to Increased Glucose Flux through Increased GLUT1 Expression in Rat Retinal Endothelial Cells

To investigate whether increased glucose flux through increased glucose transporter1 (GLUT1) expression results in increased oxidative stress and increased c-jun N-terminal kinase (JNK) phosphorylation. GLUT1-overexpressing cells were established using a rat retinal endothelial cell line. The intracellular reactive oxygen species was detected by the oxidation of 5- (and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA). Western blot was performed to determine JNK phosphorylation and lipid peroxidation. Differentially expressed genes were detected by cDNA microarray analysis and confirmed by Northern blot analysis. Clones overexpressing GLUT1 showed an approximate four- to eightfold increase in GLUT1 expression and a 44% increase in intracellular glucose concentrations. GLUT1-overexpressing cells had a 80% increase in DCF fluorescence and increased lipid peroxidation, as well as increased JNK phosphorylation. Analysis of differentially expressed genes in GLUT1-overexpressing cells showed increased expression of JNK interacting protein (JIP)-1, a scaffold protein necessary for JNK activation. Northern blot analysis confirmed upregulation of JIP-1. Immunoprecipitation showed that phosphorylated JNK, but not total JNK, coimmunoprecipitated with JIP-1 protein. At the cellular level, JIP-1 was predominantly localized in cytoplasm, especially in the perinuclear area in retinal endothelial cells. GLUT1 overexpression and increased glucose flux result in increased oxidative stress and JNK phosphorylation in immortalized rat retinal endothelial cells. Further studies are needed to understand molecular events after increased glucose flux in retinal endothelial cells and the relation between increased oxidative stress and JNK phosphorylation.

Critical Role for the Oligoadenylate Synthetase/RNase L Pathway in Response to IFN-β during Acute Ocular Herpes Simplex Virus Type 1 Infection

We previously demonstrated that IFN-beta transgene treatment protects mouse trigeminal ganglia (TG) cells from acute HSV-1 infection in vitro. However, IFN-alpha6 transgene treatment does not provide protection against acute HSV-1 infection in vitro, even though equivalent levels of IFN are expressed with both transgene treatments. In the present study we show that IFN-beta transgene treatment before acute ocular HSV-1 infection protects mice from HSV-1-mediated mortality, whereas IFN-alpha6 transgene treatment does not reduce mortality. Treatment with the IFN-beta and IFN-alpha6 transgenes was associated with increased expression of oligoadenylate synthetase (OAS)1a mRNA in the eye. However, protein kinase R mRNA was not up-regulated in the eye. In TG, only IFN-beta transgene treatment reduced infectious virus levels. Furthermore, in the absence of a functional OAS pathway, corneal HSV-1 Ag expression was more widespread, and the ability of IFN-beta transgene treatment to reduce infectious HSV-1 in eyes and TG was lost. Along with selective up-regulation of OAS1a mRNA expression in TG from IFN-beta transgene-treated mice, we found increased levels of phospho-STAT1. Likewise, p38 MAPK phosphorylation was increased in TG from IFN-beta transgene-treated mice, compared with both IFN-alpha6 and vector-treated mice. We also observed a time-dependent increase in JNK phosphorylation in TG from IFN-beta transgene-treated vs IFN-alpha6 and vector-treated mice. Our results demonstrate that IFN-beta is a potent antiviral cytokine that exerts protection against ocular HSV-1 infection via selective up-regulation of OAS1a mRNA in TG and by altering the phosphorylation of proteins in antiviral signaling cascades.