JNK Inhibitor Treatment Research Articles

ITIH4 reversed the effects of thrombin on VSMCs stiffness via JNK and ERK signaling pathway

Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.

Cadmium-induced annulus fibrosus cell senescence contributes to intervertebral disc degeneration via the JNK/p53 signaling pathway.

Investigating the impact of cadmium (Cd) on annulus fibrosus (AF) cells and its potential mechanism was the purpose of the current study. Cd was cultivated in different concentrations (0, 1, 5, 10, and 20 μM) on AF cells and the potential effects of the metal were assessed. Using the CCK-8 method, cell viability and proliferation were identified. Using transcriptome analysis, the annulus fibrosus cells were sequenced both with and without cadmium chloride. The EdU method was used to determine the rate of cell proliferation; senescence-associated β-galactosidase (SA-β-Gal) staining was used to determine the number of positive cells; and western blot, RT-PCR, and immunofluorescence were used to determine the protein and mRNA expression of senescence-associated proteins (p16, p21, and p53) and c-Jun N-terminal kinase (JNK). According to the findings, Cd has the ability to increase the production of senescence-associated genes (p16 and p21) and senescence-associated secreted phenotype (SASP), which includes IL-1β and IL-6. Through the JNK/p53 signal pathway, Cd exposure simultaneously accelerated AF cell senescence and promoted SASP. Following JNK inhibitor (SP600125) treatment, the expression of p53, JNK, and senescence-associated indices were all down-regulated. By activating the JNK/p53 signaling pathway, Cd can induce oxidative stress damage and AF cell senescence. These findings could provide a new approach for treating and preventing intervertebral disc degeneration (IVDD) caused by Cd exposure.

The Possible Connection of Two Dual Function Processes: The Relationship of Ferroptosis and the JNK Pathway.

Ferroptosis represents a typical process that has dual functions in cell fate decisions since the reduction and/or inhibition of ferroptosis is desirable for the therapies of diseases such as neurological disorders, localized ischemia-reperfusion, kidney injury, and hematological diseases, while the enhanced ferroptosis of cancer cells may benefit patients with cancer. The JNK pathway also has a real dual function in the fate of cells. Multiple factors suggest a potential link between the ferroptotic and JNK pathways; (i) both processes are ROS mediated; (ii) both can be inhibited by lipid peroxide scavengers; (iii) RAS mutations may play a role in the initiation of both pathways. We aimed to investigate the possible link between ferroptosis and the JNK pathway. Interestingly, JNK inhibitor co-treatment could enhance the cancer cytotoxic effect of the ferroptosis inducers in NRAS and KRAS mutation-harboring cells (HT-1080 and MIA PaCa-2). Since cancer’s cytotoxic effect from the JNK inhibitors could only be suspended by the ferroptosis inhibitors, and that sole JNK-inhibitor treatment did not affect cell viability, it seems that the JNK inhibitors “just” amplify the effect of the ferroptosis inducers. This cancer cell death amplifying effect of the JNK inhibitors could not be observed in other oxidative stress-driven cell deaths. Hence, it seems it is specific to ferroptosis. Finally, our results suggest that GSH content/depletion could be an important candidate for switching the anti-cancer effect of JNK inhibitors.

JNK signaling contributes to skeletal muscle wasting and protein turnover in pancreatic cancer cachexia.

Cancer cachexia patients experience significant muscle wasting, which impairs the quality of life and treatment efficacy for patients. Skeletal muscle protein turnover is imparted by increased expression of ubiquitin-proteasome pathway components. Mitogen-activated protein kinases p38 and ERK have been shown to augment E3 ubiquitin ligase expression. Utilizing reverse-phase protein arrays, we identified pancreatic cancer cell-conditioned media-induced activation of JNK signaling in myotubes differentiated from C2C12 myoblasts. Inhibition of JNK signaling with SP600125 reduced cancer cell-conditioned media-induced myotube atrophy, myosin heavy chain protein turnover, and mRNA expression of cachexia-specific ubiquitin ligases Trim63 and Fbxo32. Furthermore, utilizing an orthotopic pancreatic cancer cachexia mouse model, we demonstrated that treatment of tumor-bearing mice with SP600125 improved longitudinal measurements of forelimb grip strength. Post-necropsy measurements demonstrated that SP600125 treatment rescued body weight, carcass weight, and gastrocnemius muscle weight loss without impacting tumor growth. JNK inhibitor treatment also rescued myofiber degeneration and reduced the muscle expression of Trim63 and Fbxo32. These data demonstrate that JNK signaling contributes to muscle wasting in cancer cachexia, and its inhibition has the potential to be utilized as an anti-cachectic therapy.

PPA1 promotes NSCLC progression via a JNK- and TP53-dependent manner

Inorganic pyrophosphatase (PPA1) promotes tumor progression in several tumor types. However, the underlying mechanism remains elusive. Here, we disclosed that PPA1 expression is markedly upregulated in lung carcinoma tissue versus normal lung tissue. We also found that the non-small cell lung cancer (NSCLC) cell lines show increased PPA1 expression levels versus normal lung cell line control. Moreover, the knockdown of PPA1 promotes cell apoptosis and inhibits cell proliferation. Whereas, the ectopic expression of PPA1 reduces cell apoptosis and enhances cell proliferation. Most interestingly, the expression of mutant PPA1 (D117A) significantly abolishes PPA1-mediated effect on cell apoptosis and proliferation. The underlying mechanism demonstrated that TP53 expression deficiency or JNK inhibitor treatment could abolish PPA1-mediated NSCLC progression. In summary, the aforementioned findings in this study suggest a new pathway the PPA1 mediates NSCLC progression either via TP53 or JNK. Most important, the pyrophosphatase activity is indispensible for PPA1-mediated NSCLC progression. This may provide a promising target for NSCLC therapy.

Adaptor protein CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation

The adaptor protein CrkII is involved in several biological activities, including mitogenesis, phagocytosis, and cytoskeleton reorganization. Previously, we demonstrated that CrkII plays an important role in osteoclast differentiation and function through Rac1 activation both in vitro and in vivo. In this study, we investigated whether CrkII also regulates the differentiation and function of another type of bone cells, osteoblasts. Overexpression of CrkII in primary osteoblasts inhibited bone morphogenetic protein (BMP) 2-induced osteoblast differentiation and function, whereas knockdown of CrkII expression exerted the opposite effect. Importantly, CrkII strongly enhanced c-Jun-N-terminal kinase (JNK) phosphorylation, and the CrkII overexpression-mediated attenuation of osteoblast differentiation and function was recovered by JNK inhibitor treatment. Furthermore, transgenic mice overexpressing CrkII under control of the alpha-1 type I collagen promoter exhibited a reduced bone mass phenotype. Together, these results indicate that CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation. Given that CrkII acts as a negative and positive regulator of osteoblast and osteoclast differentiation, respectively, the regulation of CrkII expression in bone cells may help to develop new strategies to enhance bone formation and inhibit bone resorption.

P57KIP2‑mediated inhibition of human trophoblast apoptosis and promotion of invasion in vitro.

Placental hypoxia serves a role in the early stages of normal pregnancy and is involved in the pathophysiology of preeclampsia. Previously, it was suggested that p57kinase inhibitory protein (KIP)2 regulates the cell cycle during embryogenesis and apoptosis. Recent evidence has indicated that p57KIP2 is increased in preeclamptic placentas and absence of p57KIP2 induces preeclampsia‑type symptoms in rats. However, effects of p57KIP2 on apoptosis under hypoxic conditions remain to be elucidated. In the present study, HTR‑8/SVneo trophoblasts were cultured under hypoxic conditions (2% O2). Knockdown using small interfering (si)RNA and overexpression of p57KIP2 were utilized to explore the biological function of p57KIP2 in apoptosis and cell function in vitro. Furthermore, expression of p57KIP2 and apoptosis were evaluated by western blotting, flow cytometry and TUNEL assays, and the response of trophoblasts to hypoxia and the role of p57KIP2 in trophoblast migration and invasion was assessed. The role of p57KIP2 in the JNK signaling pathway in HTR‑8/SVneo trophoblasts was further studies. In vitro, protein expression of p57KIP2 was increased in HTR‑8/SVneo cells exposed to 2% O2. Exogenous p57KIP2 overexpression significantly decreased the expression of pro‑apoptosis proteins, including p53, Bax and cleaved caspase3, under hypoxic conditions for 24 h. In addition, knockdown of p57KIP2 increased the response to apoptosis following hypoxia for 24 h. The present study revealed that overexpression of p57KIP2 decreased the levels of phosphorylated‑JNK. JNK inhibitor treatment combined with the overexpression of p57KIP2 significantly decreased the levels of apoptosis and increased cell invasion and migration. Taken together, p57KIP2 knockdown significantly increased apoptosis in HTR‑8/SVneo cells exposed to 2% O2, whereas overexpression of p57KIP2 had opposite effects, mediated by the JNK/stress activated protein kinase (SAPK) signaling pathway. The results indicated that hypoxia‑induced expression of p57KIP2 promoted trophoblast migration and invasion by mediating the JNK/SAPK signaling pathway, which is crucial during placentation. These results may provide a novel molecular mechanism to understand the involvement of p57KIP2 in the pathogenesis of preeclampsia.

Abstract 153: A genome-wide CRISPR screen in hESC uncovers JNK pathway mediated inhibition of human definitive endoderm formation

Abstract Human embryonic stem cells (hESCs) are uniquely suitable for interrogating human development and birth defects with high-throughput genetic manipulation. Using CRISPR/Cas, we have conducted a forward genetic screen for genes that regulate the formation of human definitive endoderm (DE), the first step towards the generation of cells in respiratory and gastrointestinal organs. Our screen identified both known and unknown regulators of endoderm differentiation, including a previously unrecognized inhibitory mechanism mediated by the JNK/JUN pathway. JNK inhibitor treatment significantly improves the efficiency of hESC differentiation to endoderm, leading to more efficient creation of endoderm-derived pancreatic and lung lineage cells. The AP1 transcription factor JUN co-occupies ESC enhancers with SMAD2/3 and OCT4, and impedes the re-configuration of the SMAD2/3 chromatin-binding landscape during the ESC-to-DE transition. Thus the hESC-based CRISPR screen enables unbiased interrogation of the functional genome during human development, which can inform hESC directed differentiation for regenerative medicine and disease modeling. Citation Format: Qing Li, Danwei Huangfu. A genome-wide CRISPR screen in hESC uncovers JNK pathway mediated inhibition of human definitive endoderm formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 153.

Abstract B091: Disruption of cancer stemness by targeting the JNK-STAT3 pathway in neuroblastoma

Abstract Background: Neuroblastoma is one of the most challenging malignant tumors in children. The 5-year survival rate among patients with progressive tumors is still 30-40%, even after the improvement of intensive therapies. In our study, we found that JNK and its downstream factor, STAT3, are novel targets for disrupting cancer stemness in neuroblastoma. JNK is one of the major MAPKs; it has been reported that JNK or STAT3 maintains the stemness of cancer cells. We analyzed the activity of the JNK-STAT3 cascade and the effects of inhibiting JNK or STAT3 in neuroblastomas. Methods: The CHP134, IMR32, IMR5, NLF, SK-N-AS, and SHEP neuroblastoma cell lines were used in this study. The hTERT-RPE1 (RPE1) cell line was analyzed as a normal cell control model. SP600125 and JNK-IN-8 (JNK inhibitors), and Niclosamide (a STAT3 inhibitor), were used in various concentrations. A cell viability assay was performed using the CellTiter-Blue (Promega) system. The gene expression was analyzed using a THUNDERBIRD SYBR-Green real-time PCR system (TOYOBO). An immunofluorescence analysis and immunoblotting were performed using antibodies for beta-3 tubulin (SIGMA-ALDRICH), JNK, p-JNK, STAT3, p-STAT3 s727, p-STAT3 y705 (all Abcam). Results: The IMR5, NLF, SK-N-AS neuroblastoma cell lines showed remarkably lower cell viability after treatment with SP600125 or JNK-IN-8, in comparison to nontreated cells. Treatment with JNK inhibitors led to growth retardation in RPE1 cells; however, this cell line showed differentiation rather than cell death. Cell death was more significant in cells with faster proliferation than in cells with slower proliferation, which showed differentiation after JNK inhibitor treatment. The phosphorylation of STAT3 serine 727 (s727), but not tyrosine 705 (y705), corresponded well to the JNK expression in neuroblastoma cells; cells with MYCN amplification or 1p deletion, both unfavorable prognostic markers, showed high levels of JNK expression and strong STAT3 s727 phosphorylation, while STAT3 y705 phosphorylation was not found in any cell lines. JNK inhibitor treatment resulted in a dose-dependent decrease in STAT3 s727 phosphorylation in IMR5 cells. The STAT3 inhibitor nicrosamide induced cell death at high concentrations and cell differentiation at low concentrations. The expression of CXCR4, a cancer stem cell marker, was downregulated by JNK or STAT3 inhibitors. Discussion: Targeting the JNK-STAT3 cascade induced both cell death and cell differentiation. Our results suggest that JNK-STAT3 maintains cancer stemness, and that the inhibition of this pathway disrupts the stemness in neuroblastomas. The JNK-STAT3 cascade is thus considered to be a novel target for neuroblastoma therapy. Citation Format: Mayumi Higashi, Kiyokazu Kin, Tatsuro Tajiri. Disruption of cancer stemness by targeting the JNK-STAT3 pathway in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B091.

Treatment with a JNK inhibitor increases, whereas treatment with a p38 inhibitor decreases, H2O2-induced calf pulmonary arterial endothelial cell death.

Oxidative stress induces apoptosis in endothelial cells (ECs). Reactive oxygen species (ROS) promote cell death by regulating the activity of various mitogen-activated protein kinases (MAPKs) in ECs. The present study investigated the effects of MAPK inhibitors on cell survival and glutathione (GSH) levels upon H2O2 treatment in calf pulmonary artery ECs (CPAECs). H2O2 treatment inhibited the growth and induced the death of CPAECs, as well as causing GSH depletion and the loss of mitochondrial membrane potential (MMP). While treatment with the MEK or JNK inhibitor impaired the growth of H2O2-treated CPAECs, treatment with the p38 inhibitor attenuated this inhibition of growth. Additionally, JNK inhibitor treatment increased the proportion of sub-G1 phase cells in H2O2-treated CPAECs and further decreased the MMP. However, treatment with a p38 inhibitor reversed the effects of H2O2 treatment on cell growth and the MMP. Similarly, JNK inhibitor treatment further increased, whereas p38 inhibitor treatment decreased, the proportion of GSH-depleted cells in H2O2-treated CPAECs. Each of the MAPK inhibitors affected cell survival, and ROS or GSH levels differently in H2O2-untreated, control CPAECs. The data suggest that the exposure of CPAECs to H2O2 caused the cell growth inhibition and cell death through GSH depletion. Furthermore, JNK inhibitor treatment further enhanced, whereas p38 inhibitors attenuated, these effects. Thus, the results of the present study suggest a specific protective role for the p38 inhibitor, and not the JNK inhibitor, against H2O2-induced cell growth inhibition and cell death.

Enhancement of radiosensitivity by inhibition of c-Jun N-terminal kinase activity in a Lewis lung carcinoma‑bearing subcutaneous tumor mouse model.

Stereotactic radiosurgery has been recognized as an effective treatment approach for metastatic brain tumors. By increasing the sensitivity of the tumor to radiation and decreasing the marginal dose, it is possible to improve therapeutic efficacy and decrease side-effects. In radiation-induced cells, c-Jun N-terminal kinase (JNK) signaling mediates the phosphorylation of H2AX, which indicates DNA damage sensitivity and modulates the effect of radiation. Lewis lung cancer (LLC) and breast cancer (4T1) cells were irradiated with a Gamma Knife in cell culture tubes. To evaluate the relationship between radiosensitivity and JNK activity, clonogenic assay was performed. DNA damage response was estimated by γH2AX focus formation assay and apoptosis‑related protein levels were assessed by western blotting. The mice were subcutaneously inoculated with LLC cells, and irradiated concomitantly with JNK inhibitor treatment. The effect of the JNK inhibitor was investigated by tumor volumetry and immunohistochemistry. γH2AX expression, which mediates repair of radiation‑induced DNA damage, was reduced in the cancer cell group pretreated with the JNK inhibitor. This finding shows that JNK inhibition may increase the radiosensitivity in radiated lung and breast cancer cells. For the invivo study, irradiated tumor growth was significantly delayed in the JNK inhibitor-treated mouse group. Blockade of JNK signaling decreased γH2AX expression and increased apoptosis in the radiation-induced cancer cells. JNK inhibitor may be useful for enhancing the radiosensitivity of lung and breast cancer cells and improving the treatment efficacy of radiosurgical approaches for metastatic brain tumors.

Time-staggered inhibition of JNK effectively sensitizes chemoresistant ovarian cancer cells to cisplatin and paclitaxel.

Ovarian cancer is the most lethal gynecological malignancy, for which platinum- and taxane-based chemotherapy plays a major role. Chemoresistance of ovarian cancer poses a major obstacle to the successful management of this devastating disease; however, effective measures to overcome platinum and taxane resistance are yet to be established. In the present study, while investigating the mechanism underlying the chemoresistance of ovarian cancer, we found that JNK may have a key role in the resistance of ovarian cancer cells to cisplatin and paclitaxel. Importantly, whereas simultaneous application of a JNK inhibitor and either of the chemotherapeutic agents had contrasting effects for cisplatin (enhanced cytotoxicity) and paclitaxel (decreased cytotoxicity), JNK inhibitor treatment prior to chemotherapeutic agent application invariably enhanced the cytotoxicity of both drugs, suggesting that the basal JNK activity is commonly involved in the chemoresistance of ovarian cancer cells to cisplatin and paclitaxel in contrast to drug‑induced JNK activity which may have different roles for these two drugs. Furthermore, we confirmed using non-transformed human and rodent fibroblasts that sequential application of the JNK inhibitor and the chemotherapeutic agents did not augment their toxicity. Thus, our findings highlight for the first time the possible differential roles of the basal and induced JNK activities in the chemoresistance of ovarian cancer cells and also suggest that time‑staggered JNK inhibition may be a rational and promising strategy to overcome the resistance of ovarian cancer to platinum- and taxane-based chemotherapy.

EphrinB1 Interacts with CNK1 and Promotes Cell Migration through c-Jun N-terminal Kinase (JNK) Activation

The Eph receptors and their membrane-bound ligands, ephrins, play important roles in various biological processes such as cell adhesion and movement. The transmembrane ephrinBs transduce reverse signaling in a tyrosine phosphorylation-dependent or -independent, as well as PDZ-dependent manner. Here, we show that ephrinB1 interacts with Connector Enhancer of KSR1 (CNK1) in an EphB receptor-independent manner. In cultured cells, cotransfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1-mediated JNK activation is reduced by overexpression of dominant-negative RhoA. Overexpression of CNK1 alone is sufficient for activation of RhoA; however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-immunoprecipitation data showed that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA and JNK signaling components, such as p115RhoGEF and MKK4. Furthermore, adhesion to fibronectin or active Src overexpression increases ephrinB1/CNK1 binding, whereas blocking Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. EphrinB1 overexpression increases cell motility, however, CNK1 depletion by siRNA abrogates ephrinB1-mediated cell migration and JNK activation. Moreover, Rho kinase inhibitor or JNK inhibitor treatment suppresses ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 is required for ephrinB1-induced JNK activation and cell migration.

Induction of IL-12 production by the activation of discoidin domain receptor 2 via NF-κB and JNK pathway

We investigated the mechanism involving discoidin domain receptor 2 (DDR2) mediated production of interleukin 12 (IL-12). When compared to control, collagen I upregulated the IL-12 luciferase activity on DDR2 expressing cells. Collagen I induced the phosphorylation of DDR2 and enhanced the phosphorylation of mitogen activated protein kinase (MAPK) kinases. In addition, NF-κB binding activity was enhanced when the cells expressing NF-κB reporter were exposed to collagen I. Moreover, when IL-12 reporter transfected cells were treated with biochemical inhibitors of c-Jun N-terminal kinase (JNK) and NF-κB, collagen-induced IL-12 promoter activity was significantly downregulated in comparison to non-treated cells. Similarly, confirmatory experiments on murine dendritic cells revealed that IL-12 promoter activity is dose dependently downregulated upon NF-κB and JNK inhibitor treatment on collagen I stimulation. In summary, DDR2 is involved in the collagen I-induced IL-12 production via NF-κB and JNK pathway.

C-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic proteins 2 (BMP2). Mitogen-activated protein kinase (MAPK) cascades, such as extracellular signal-regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun-N-terminal kinase (JNK), the other kinase of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first showed that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3-E1 cell lines. We then showed that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss-of-function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN-JNK1). Consistently, BMP2-induced osteoblastic differentiation is reduced by JNK1 gain-of-function, such as enforced expression of a constitutively active JNK1 (CA-JNK1). Most importantly, we showed that Runx2 is required for JNK1-mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3-E1 cells to the extent similar to that achieved by overexpression of wild-type (WT) Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2-induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation.

CJUN N-terminal kinase (JNK) activation mediates islet amyloid-induced beta cell apoptosis in cultured human islet amyloid polypeptide transgenic mouse islets.

Aggregation of human islet amyloid polypeptide (hIAPP) as islet amyloid is associated with increased beta cell apoptosis and reduced beta cell mass in type 2 diabetes. Islet amyloid formation induces oxidative stress, which contributes to beta cell apoptosis. The cJUN N-terminal kinase (JNK) pathway is a critical mediator of beta cell apoptosis in response to stress stimuli including oxidative stress and exogenous application of hIAPP. We determined whether amyloid formation by endogenous hIAPP mediates beta cell apoptosis through JNK activation and downstream signalling pathways. hIAPP transgenic and non-transgenic mouse islets were cultured for up to 144 h in 16.7 mmol/l glucose to induce islet amyloid in the presence or absence of the amyloid inhibitor Congo Red or a cell-permeable JNK inhibitor. Amyloid, beta cell apoptosis, JNK signalling and activation of downstream targets in the intrinsic and extrinsic apoptotic pathways were measured. JNK activation occurred with islet amyloid formation in hIAPP transgenic islets after 48 and 144 h in culture. Neither high glucose nor the hIAPP transgene alone was sufficient to activate JNK independent of islet amyloid. Inhibition of islet amyloid formation with Congo Red reduced beta cell apoptosis and partially decreased JNK activation. JNK inhibitor treatment reduced beta cell apoptosis without affecting islet amyloid. Islet amyloid increased mRNA levels of markers of the extrinsic (Fas, Fadd) and intrinsic (Bim [also known as Bcl2l11]) apoptotic pathways, caspase 3 and the anti-apoptotic molecule Bclxl (also known as Bcl2l1) in a JNK-dependent manner. Islet amyloid formation induces JNK activation, which upregulates predominantly pro-apoptotic signals in both extrinsic and intrinsic pathways, resulting in beta cell apoptosis.

The inflammatory cytokine tumor necrosis factor modulates the expression of Salmonella typhimurium effector proteins

Tumor necrosis factor α (TNF-α)is a host inflammatory factor. Bacteria increase TNF-α expression in a variety of human diseases including infectious diseases, inflammatory bowel diseases, and cancer. It is unknown, however, how TNF-α directly modulates bacterial protein expression during intestinal infection and chronic inflammation. In the current study, we hypothesize that Salmonella typhimurium senses TNF-α and show that TNF-α treatment modulates Salmonella virulent proteins (called effectors), thus changing the host-bacterial interaction in intestinal epithelial cells. We investigated the expression of 23 Salmonella effectors after TNF-α exposure. We found that TNF-α treatment led to differential effector expression: effector SipA was increased by TNF-α treatment, whereas the expression levels of other effectors, including gogB and spvB, decreased in the presence of TNF-α. We verified the protein expression of Salmonella effectors AvrA and SipA by Western blots. Furthermore, we used intestinal epithelial cells as our experimental model to explore the response of human intestinal cells to TNF-α pretreated Salmonella. More bacterial invasion was found in host cells colonized with Salmonella strains pretreated with TNF-α compared to Salmonella without TNF-α treatment. TNF-α pretreated Salmonella induced higher proinflammatory JNK signalling responses compared to the Salmonella strains without TNF-α exposure. Exposure to TNF-α made Salmonella to induce more inflammatory cytokine IL-8 in intestinal epithelial cells. JNK inhibitor treatment was able to suppress the effects of TNF-pretreated-Salmonella in enhancing expressions of phosphorylated-JNK and c-jun and secretion of IL-8. Overall, our study provides new insights into Salmonella-host interactions in intestinal inflammation.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.