Increased JNK Activity Research Articles

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Toxic effects of mutant huntingtin in axons are mediated by its proline-rich domain.

Huntington's disease results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mHTT to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by c-Jun N-terminal kinases (JNKs) and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNKs. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem Huntington's disease patients. Together, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in Huntington's disease.

Abstract P1-08-22: The role of DAXX as a critical growth regulator of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous disease that frequently presents at a metastatic stage or upon initial early stage diagnosis, and may recur quickly. Treatment options are limited outside of cytotoxic chemotherapy in the adjuvant or metastatic setting and there is an immediate need to identify and validate novel biomarkers that predict sensitivity to chemotherapy. Previously, it was shown that the pro-apoptotic protein DAXX is required for paclitaxel-mediated breast cancer cell death. Based on these data and our recent discoveries that DAXX is a potent inhibitor of breast cancer stem cells in ER+ breast cancer (Peiffer et al., Cancer Res. 2019; Peiffer et al., NPJ Breast Cancer. 2020), we next explored the role of DAXX in TNBC. We found that high DAXX expression in TNBC cells regulates cell proliferation and activities of PARP-1 and JNK; two pathways required for cell death. The goals of the current work were to investigate the mechanism by which DAXX regulates proliferation at the level of PARP-1 and/or JNK and test if DAXX expression predicts sensitivity to carboplatin, paclitaxel, doxorubicin, the PARP-1 inhibitor olaparib, or the JNK inhibitor SP600125 in TNBC cells. Methods: Proliferation and cell cycle analysis were performed in three TNBC cell lines (MDA-MB-231, BT549, and MDA-MB-468) under DAXX expression and depletion conditions using an siRNA approach. Sensitivity to carboplatin, paclitaxel, and doxorubicin was determined by measuring proliferation in a dose-dependent manner. PARP-1 activity was assessed by detecting global protein PARylation levels using Western blotting. JNK activation was analyzed by measuring phosphorylated JNK levels. A JNK inhibitor (SP600125) was used to determine if JNK signaling was responsible for restricting cell proliferation and if DAXX was required for the elevated JNK activity. Olaparib was used to assess the role of PARP-1 activation in DAXX-depleted cells. Results: The TNBC cell lines expressed high levels of DAXX. Depletion of DAXX increased cell proliferation by promoting cell cycle progression through the S-phase. The consequence of this increased cell proliferation was decreased sensitivity to carboplatin and paclitaxel, but not to doxorubicin. Mechanistically, at least one chemotherapeutic agent, carboplatin induced JNK activation and DAXX was required for the increased JNK activity. The JNK inhibitor (SP600125) partially reversed resistance to carboplatin in DAXX-depleted cells. Additionally, genetic knockdown of DAXX resulted in elevated PARP-1 activity as measured by global PARylation of proteins. PARP-1 blockade using olaparib also partially reversed resistance to carboplatin in DAXX-depleted cells. Conclusions: These results suggest that DAXX is a critical growth regulator and predictor of response to carboplatin and paclitaxel in triple negative breast cancer cells. DAXX is necessary to activate the JNK pathway in response to carboplatin and it is this carboplatin-mediated JNK signaling that inhibits cell proliferation. Furthermore, DAXX limits PARP-1 activity and is necessary for increased sensitivity to carboplatin or paclitaxel. Data herein support assessing DAXX expression in triple negative tumors to determine if high DAXX expression predicts increased sensitivity to carboplatin, paclitaxel, and if DAXX-low expressing TNBC cells require addition of a JNK or PARP-1 inhibitor. Citation Format: Clodia Osipo, Debra Wyatt, Michelle Fernandez, Daniel S Peiffer, Kathy S Albain. The role of DAXX as a critical growth regulator of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-22.

Nicotinamide riboside kinase-2 inhibits JNK pathway and limits dilated cardiomyopathy in mice with chronic pressure overload

Nicotinamide riboside kinase-2 (NRK-2) has recently emerged as a critical regulator of cardiac remodeling however, underlying molecular mechanisms is largely unknown. To explore the same, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant deterioration during the maladaptive cardiac remodeling phase. Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure (HF) reflected by morphometric parameters as well as increased fetal genes, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy (CM) and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing in AC16 cardiomyocytes displayed significantly attenuated fetal genes ANP and BNP expression. Consistently, NRK-2 overexpression attenuated angiotensin II (Ang II)-induced cardiomyocyte death. Mechanistically, we identified NRK-2 as a regulator of c-jun N-terminal kinase (JNK) MAP kinase and mitochondrial function where NRK-2 overexpression in human cardiomyocytes markedly suppressed the Ang II-induced JNK activation and mitochondrial depolarization. Thus, our results demonstrate that NRK-2 plays protective roles in pressure overload (PO)-induced dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy (DCM), interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation and mitochondrial dysfunction.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract P326: Nicotinamide Riboside Kinase-2 Deficiency Promotes Pressure Overload- Induced Dilated Cardiomyopathy And Heart Failure

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with a poor prognosis. Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin-binding protein, is predominantly expressed in skeletal muscle and upregulates in several heart failure models. Emerging pieces of evidence suggest that NRK-2 plays a key role in cardiac pathogenesis, however, its role in chronic pressure overload (PO)-induced cardiac remodeling is largely unknown. To investigate the potential role of NRK-2 in PO-induced DCM and delineate the underlying molecular mechanisms, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant LV chamber dilatation [LVIDd(mm); 5.32±0.36 vs. 4.75±0.39, P =0.001] and functional deterioration [LVEF(%); 20.53±8.81vs. 31.2±7.0, P =0.003] during the maladaptive cardiac remodeling phase (6 week). Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes ANP and BNP expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing AC16 cardiomyocytes displayed significantly attenuated fetal genes expression and, NRK-2 further suppressed the fetal gene expression when challenged with angiotensin II. Consistently, NRK-2 overexpression attenuated angiotensin II-induced cardiomyocyte death. Mechanistically, NRK-2 was identified as a critical regulator of JNK MAP kinase and NRK-2 overexpression markedly suppressed the angiotensin II-induced JNK activation. Overall, our results demonstrate that NRK-2 regulates dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.

Abstract 1085: PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells

Abstract Background Colorectal cancers (CRCs) are intrinsically drug resistant tumors with frequent low or absent tumor cell PD-L1 expression. Previously, we reported that PD-L1 depletion in CRC cells can confer chemoresistance. Given that JNK signaling has been implicated in cell survival and drug resistance in CRC cells, we examined the role of JNK and its regulation in chemoresistance in CRC cells with loss of PD-L1. Materials &amp; MethodsHuman CRC cell lines (RKO, DLD1 and HCT15) were utilized and parental and PD-L1 knockout/knockdown cells were generated. Cells were treated with a JNK1-3 inhibitor (JNK-IN-8) alone or combined with a MEK1/2 inhibitor (cobimetinib), CPT-11 or oxaliplatin. Protein/protein interaction was analyzed by immunoprecipitation (IP); protein/RNA interaction was analyzed by RNA IP assay. RNA level and half life were measured by QRT-PCR. Apoptosis was measured by cleaved caspase-3 and quantified by Annexin V labeling using FACS. Colony formation assay was performed. ResultsPD-L1 depletion in CRC cells was shown to enhance JNK activity that was due to reduced mRNA stability of the upstream CYLD deubiquitinase. Since PD-L1 competes with the ribonuclease EXOSC10 for binding to CYLD mRNA, loss of PD-L1 increases the interaction of EXOSC10 and CYLD mRNA resulting in its ribonuclease-mediated degradation and activation of JNK signaling. JNK activation increased BIM phosphorylation at Thr116 that promotes its sequestration by MCL-1 and BCL-2. Notably, a selective and irreversible JNK1-3 inhibitor (JNK-IN-8) reduces BIMThr116 phosphorylation and can release BIM from its sequestration by MCL-1 and BCL-2 to promote apoptosis. Furthermore, use of JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination in tumor cells lacking PD-L1 is shown to promote apoptosis and reduce long-term clonogenic survival induced by multiple anti-cancer drugs. As confirmation, knockdown of PD-L1 can confer drug resistance in a human colon cancer-derived organoid model that can be reversed by JNK-IN-8. Conclusion Tumor cells with low or absent PD-L1 expression show increased JNK activity that promotes BIM sequestration by MCL-1/BCL-2 to confer multiple drug resistance that can be reversed by a JNK inhibitor. Citation Format: Frank A. Sinicrope, Lei Sun, Arpad Patai, Tara L. Hogenson, Martin E. Fernandez-Zapico, Bo Qin. PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1085.

Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

Acetaminophen (APAP)-induced liver necrosis is a form of regulated cell death (RCD) in which APAP activates the mitogen-activated protein kinases (MAPKs) and specifically the c-Jun-N-terminal kinase (JNK) pathway, leading to necrotic cell death. Previously, we have shown that receptor interacting protein kinase-1 (RIPK1) knockdown is also protective against APAP RCD upstream of JNK. However, whether the kinase or platform function of RIPK1 is involved in APAP RCD is not known. To answer this question, we used genetic mouse models of targeted hepatocyte RIPK1 knockout (RIPK1HepCKO) or kinase dead knock-in (RIPK1D138N) and adult hepatocyte specific knockout of the cytoprotective protein A20 (A20HepCKO), known to interact with RIPK1, to study its potential involvement in MAPK signaling. We observed no difference in injury between WT and RIPK1D138N mice post APAP. However, RIPK1HepCKO was protective. We found that RIPK1HepCKO mice had attenuated pJNK activation, while A20 was simultaneously upregulated. Conversely, A20HepCKO markedly worsened liver injury from APAP. Mechanistically, we observed a significant upregulation of apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation in A20HepCKO mice compared with littermate controls. We also demonstrated that A20 coimmunoprecipitated (co-IP) with both RIPK1 and ASK1, and that in the presence of RIPK1, there was less A20-ASK1 association than in its absence. We conclude that the kinase-independent platform function of RIPK1 is involved in APAP toxicity. Adult RIPK1HepCKO mice are protected against APAP by upregulating A20 and attenuating JNK signaling through ASK1, conversely, A20HepCKO worsens injury from APAP.

Thrombospondin-1 Receptor CD47 Overexpression Contributes to P-Glycoprotein-Mediated Multidrug Resistance Against Doxorubicin in Thyroid Carcinoma FTC-133 Cells.

It is now admitted that in addition to acquired resistance, the tumor microenvironment contributes to the development of chemo-resistance and malignant progression. In a previous study, we showed that Dox induced apoptosis in FTC-133 cells by trigging JNK pathway. This process was accompanied by a decrease of thrombospondin-1 (TSP-1) expression. Moreover, exogenous TSP-1 or its C-terminal-derived peptide interact with receptor CD47 and are able to protect FTC-133 cells against Dox-induced apoptosis. Here, we investigated the involvement of TSP-1/CD47 interaction in a context of acquired multidrug resistance in FTC-133 cells. To that end, we established a Dox-resistant cell line (FTC-133R cells) which developed a resistance against Dox-induced apoptosis. Cell viability was evaluated by Uptiblue assay, nuclear Dox was measured by microspectrofluorimetry, caspase activity was measured by fluorescence of cleaved caspase-3 substrate, gene expression was evaluated by RT-PCR and protein expression was examined by western-blot. Our results showed that FTC-133R overexpressed the P-gp and were 15-fold resistant to Dox. JNK phosphorylation and Dox-induced apoptosis were reduced in FTC-133R cells. Expression of CD47 was increased in FTC-133R cells but TSP-1 expression presented similar levels in two cell lines. VPL restored Dox nuclear uptake and FTC-133R cell sensitivity to apoptosis and induced a decrease in CD47 mRNA expression. Moreover, knockdown of CD47 in FTC-133R cells induced an increase in JNK activation and sensitized FTC-133R cells to Dox. Our data suggest that CD47 is able to contribute to the protection of FTC-133R cells against Dox-induced apoptosis and/or to potentiate the acquired Dox resistance.

Peroxiredoxin 6 mediates acetaminophen-induced hepatocyte death through JNK activation

Acetaminophen (APAP) is one of the most frequently used drugs; however, its overdose leads to acute liver injury. Recently, studies have reported that the adduction of peroxiredoxin 6 (PRDX6), a member of the PRDX family of antioxidant enzymes, is associated with liver diseases. However, the role of PRDX6 in APAP-induced liver injury remains unclear. Here, we assessed both age-matched (about 12 weeks) PRDX6-overexpressing transgenic mice (PRDX6 mice) and wild type (WT) mice presenting acute liver injury induced by the intraperitoneal injection of APAP (500 mg/kg). Although PRDX6 is known as an antioxidant enzyme, PRDX6 mice unexpectedly demonstrated severe liver injury following APAP injection compared with WT mice. We observed that PRDX6 was hyperoxidized after APAP administration. Additionally, calcium-independent phospholipase A2 (iPLA2) activity and lysophosphatidylcholine (LPC) levels were markedly elevated in PRDX6 mice following APAP administration. Moreover, APAP-induced JNK phosphorylation was considerably increased in the liver of PRDX6 mice. MJ33, an inhibitor of PRDX6, attenuated APAP-induced liver injury both in WT and PRDX6 mice. Notably, MJ33 reduced the APAP-induced increase in JNK activation, iPLA2 activity, and LPC levels. Although SP600125, a JNK inhibitor, abolished APAP-induced liver injury, it failed to affect the APAP-induced hyperoxidation of PRDX6, iPLA2 activity, and LPC levels. These results suggested that PRDX6 was converted to the hyperoxidized form by the APAP-induced high concentration of hydrogen peroxides. In the liver, hyperoxidized PRDX6 induced cellular toxicity via JNK activation by enhancing iPLA2 activity and LPC levels; this mechanism appears to be a one-way cascade.

Contribution of Increased Expression of Yin Yang 2 to Development of Cardiomyopathy.

Yin Yang 2 (YY2) is a member of the Yin Yang family of transcription factors. Although the bioactivity of YY2 has been previously studied, its role in cardiovascular diseases is not known. We observed the increased expression of YY2 in failing human hearts compared with control hearts, raising the question of whether YY2 is involved in the pathogenesis of cardiomyopathy. To investigate the potential contribution of YY2 to the development of cardiomyopathy, we crossed two independent transgenic (Tg) mouse lines, pCAG-YY2-Tg+and alpha-myosin heavy chain-cre (α-MHC-Cre), to generate two independent double transgenic (dTg) mouse lines in which the conditional cardiomyocyte-specific expression of YY2 driven by the α-MHC promoter was mediated by Cre recombinase, starting at embryonic day 9.0. In dTg mice, we observed partial embryonic lethality and hearts with defective cardiomyocyte proliferation. Surviving dTg mice from both lines developed cardiomyopathy and heart failure that occurred with aging, showing different degrees of severity that were associated with the level of transgene expression. The development of cardiomyopathy was accompanied by increased levels of cardiac disease markers, apoptosis, and cardiac fibrosis. Our studies further revealed that the Cre-mediated cardiomyocyte-specific increase in YY2 expression led to increased levels of Beclin 1 and LC3II, indicating that YY2 is involved in mediating autophagic activity in mouse hearts in vivo. Also, compared with control hearts, dTg mouse hearts showed increased JNK activity. Because autophagy and JNK activity are important for maintaining cardiac homeostasis, the dysregulation of these signaling pathways may contribute to YY2-induced cardiomyopathy and heart failure in vivo.

Amino acid modified gadofullerene protects against insulin resistance induced by oxidative stress in 3T3-L1 adipocytes.

Reactive oxygen species (ROS) have been shown to be involved in the development of insulin resistance; thus, antioxidant therapy is a promising strategy in the treatment of insulin resistance related diseases. In this study, we synthesized a high-efficiency ROS scavenger, an amino acid derivative of Gd@C82 (GF-Ala), and evaluated its effects on ROS-mediated insulin resistance in 3T3-L1 adipocytes. The exposure of adipocytes to H2O2 (generated by glucose oxidase) resulted in an elevated level of ROS, a reduced content of GSH and an increase in JNK activation, concomitantly with a decrease in insulin-stimulated PI3K, Akt and p70S6K activation and cellular glucose uptake. The pre-treatment of cells with GF-Ala suppressed H2O2-induced abnormalities in the cellular redox state and insulin signalling pathway and attenuated insulin resistance in a dose-dependent manner. Altogether, gadofullerene has potential therapeutic prospects in the prevention and treatment of insulin resistance related diseases.

Inhibition of JNK ameliorates depressive-like behaviors and reduces the activation of pro-inflammatory cytokines and the phosphorylation of glucocorticoid receptors at serine 246 induced by neuroinflammation

Depression is associated with immune dysregulation and the aberrant activity of the hypothalamic-pituitary-adrenal (HPA) axis. However, the neurobiological molecular mechanisms underlying these associations remain unclear. c-Jun amino-terminal kinase (JNK), an important modulator in inflammation and stress responses, is often critically implicated in the development of central nervous system diseases. However, whether and how JNK mediates neuroinflammation-induced depression remains largely unknown. In this study, we investigated the role of JNK in depressive-like behaviors induced by central lipopolysaccharide (LPS) infusion. The results showed that LPS infusion led to depressive-like behaviors, accompanied by increased proinflammatory cytokine expression, increased JNK activation, and upregulated glucocorticoid receptor (GR) phosphorylation at serine 246 (pGR-Ser246) in the habenula (Hb), amygdala (Amyg) and medial prefrontal cortex (mPFC). Treatment with SP600125, a known JNK inhibitor, prevented the LPS-induced hyper-activation of JNK and alleviated depressive-like behaviors. Moreover, LPS-induced increases in the expression levels of TNF-α, IL-1β and pGR-Ser246 in these brain regions were reduced when the rats were treated with SP600125. Our results show, for the first time, that JNK activities in the Hb, Amyg, and mPFC are involved in the modulation of neuroinflammation-induced depression and participate in the regulation of the expression of proinflammatory cytokines and GR phosphorylation, which are pathological factors associated with depression. Our findings provide new insights into the mechanism of neuroinflammation-associated depression and suggest that the JNK pathway may be a potential target for treating inflammation-related depression.

Proteomic alteration of porcine intestinal epithelial cells after pretreatment with Lactobacillus plantarum followed by infection with enterotoxigenic Escherichia coli F4

Enterotoxigenic Escherichia coli (ETEC) F4 causes diarrhea in infants and weaned piglets. The technique of isobaric tags for relative and absolute quantitation (iTRAQ) was used in this study to determine the differentially expressed proteins in porcine intestinal epithelial cells (IPEC-J2) after pretreatment with Lactobacillus plantarum (LP) followed by challenge with ETEC F4. A total of 4771 proteins were identified in IPEC-J2 cells, with 90, 105, and 134 differentially expressed proteins in cells exposed to ETEC, LP, and LP + ETEC, respectively. The COG analysis divided the identified proteins into 20 categories. The GO and KEGG annotation indicated that most of the differentially expressed proteins were enriched in various biological metabolism including cell cycle control, cell division and differentiation. Additionally, western blotting analyses confirmed the reduced abundance of selected proteins of the mTOR and MAPK signal pathways affected by ETEC F4. Moreover, LP pretreatment increased JNK activation in IPEC-J2 cells infected with ETEC F4. These results may provide further insights into the mechanisms involved in the interaction between ETEC F4 and intestinal epithelial cells, and broaden the understanding of the protective effects of LP in alleviating ETEC-provoked diarrhea of piglets.

Cofilin Phosphorylation Mediates Proliferation in Response to Platelet-Derived Growth Factor-BB in Rat Aortic Smooth Muscle Cells

Cofilin, an actin-binding protein, is essential for a variety of cell responses. In this study, we investigated the correlation between proliferation and cofilin phosphorylation in response to platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells (RASMCs). The phosphorylation of cofilin and activity of mitogen-activated protein kinase (MAPK) were measured by Western analyses and proliferation in RASMCs was measured by BrdU incorporation assays. The phosphorylation of cofilin in RASMCs was decreased by PDGF-BB treatment at 10 min, but recovered to the level of the quiescent state at 60 min. PDGF-BB–induced dephosphorylation of cofilin was inhibited by pretreatment with piceatannol (a spleen tyrosine kinase [Syk] inhibitor), PP2 (a Src inhibitor), or SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor), but not by PD98059, an inhibitor of extracellular signal-regulated kinase 1 /2. PDGF-BB increased JNK activity and proliferation, and these responses were suppressed by kinase inhibitors and small interference RNA-cofilin. The results suggest that PDGF-BB–induced dephosphorylation of cofilin can be promoted via the JNK pathway, which is regulated by both Syk and Src kinases and that cofilin dephosphorylation may be involved in PDGF-BB–induced RASMC proliferation.

RNAi-mediated knockdown of DJ-1 leads to mitochondrial dysfunction via Akt/GSK-3ß and JNK signaling pathways in dopaminergic neuron-like cells

Deletions or some mutations in the gene encoding the multifunctional protein, DJ-1, have been considered to be linked with autosomal recessive early onset Parkinson’s disease (PD). Current emerging evidence suggests that DJ-1 is involved in the protection against oxidative stress-induced mitochondrial damage. However, the exact molecular mechanisms underlying this are not completely clear. The aim of this study was to investigate the effects of DJ-1 on the Akt pathway, nuclear factor erythroid 2-related factor (Nrf2), and c-Jun N-terminal kinase (JNK) with regard to modulating mitochondrial function. Here we showed that knockdown of DJ-1 resulted in mitochondrial dysfunction, including a decrease in active mitochondrial mass, complex I deficits, and inhibition of cellular adenosine 5′-triphosphate (ATP) content in the dopaminergic neuron-like cells PC12 and SH-SY5Y. Additionally, loss of DJ-1 impaired Akt signaling, and reduced nuclear translocation of Nrf2, thereby inhibiting activity of Nrf2-regulated downstream antioxidant enzymes such as heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. Moreover, DJ-1 knockdown also led to a significant increase in the mitochondrial reactive oxygen species, and then promoted the activation of JNK pathways. Furthermore, oxidative stress and mitochondrial dysfunction induced by knockdown of DJ-1 were blocked by a JNK inhibitor, which confirmed the important role of JNK activation in mitochondrial dysfunction. In conclusion, the present study indicates that DJ-1 knockdown leads to mitochondrial dysfunction in dopaminergic neuron-like cells, at least in part, through suppressing the Akt/GSK3β pathway and impairing the oxidative stress response, as well as through the subsequent increased JNK activation in dopaminergic neuron-like cells.

Impact of hepatic HSP72 on insulin signaling.

Heat shock protein 72 (HSP72) is a major inducible molecule in the heat shock response that enhances intracellular stress tolerance. Decreased expression of HSP72 is observed in type 2 diabetes, which may contribute to the development of insulin resistance and chronic inflammation. We used HSP72 knockout (HSP72-KO) mice to investigate the impact of HSP72 on glucose metabolism and endoplasmic reticulum (ER) stress, particularly in the liver. Under a high-fat diet (HFD) condition, HSP72-KO mice showed glucose intolerance, insulin resistance, impaired insulin secretion, and enhanced hepatic gluconeogenic activity. Furthermore, activity of the c-Jun NH2-terminal kinase (JNK) was increased and insulin signaling suppressed in the liver. Liver-specific expression of HSP72 by lentivirus (lenti) in HFD-fed HSP72-KO mice ameliorated insulin resistance and hepatic gluconeogenic activity. Furthermore, increased adipocyte size and hepatic steatosis induced by the HFD were suppressed in HSP72-KO lenti-HSP72 mice. Increased JNK activity and ER stress upon HFD were suppressed in the liver as well as the white adipose tissue of HSP72-KO lenti-HSP72 mice. Thus, HSP72 KO caused a deterioration in glucose metabolism, hepatic gluconeogenic activity, and β-cell function. Moreover, liver-specific recovery of HSP72 restored glucose homeostasis. Therefore, hepatic HSP72 may play a critical role in the pathogenesis of type 2 diabetes.

Neuronal MAP kinase p38\u03b1 inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

Modulation of behavioural responses by neuronal signalling pathways remains incompletely understood. Signalling via mitogen-activated protein (MAP) kinase cascades regulates multiple neuronal functions. Here, we show that neuronal p38α, a MAP kinase of the p38 kinase family, has a critical and specific role in modulating anxiety-related behaviour in mice. Neuron-specific p38α-knockout mice show increased levels of anxiety in behaviour tests, yet no other behavioural, cognitive or motor deficits. Using CRISPR-mediated deletion of p38α in cells, we show that p38α inhibits c-Jun N-terminal kinase (JNK) activity, a function that is specific to p38α over other p38 kinases. Consistently, brains of neuron-specific p38α-knockout mice show increased JNK activity. Inhibiting JNK using a specific blood-brain barrier-permeable inhibitor reduces JNK activity in brains of p38α-knockout mice to physiological levels and reverts anxiety behaviour. Thus, our results suggest that neuronal p38α negatively regulates JNK activity that is required for specific modulation of anxiety-related behaviour.

Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer to Radiation Therapy through Oxidative Activation of the JNK Apoptotic Pathway.

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. Cerium oxide nanoparticles (CONPs) are currently being tested in pre-clinical trials as an adjuvant to sensitize pancreatic cancer cells to RT and protect normal tissues from the harmful side effects. CONPs were not able to significantly affect RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, activation of c-Jun terminal kinase (JNK), a key driver of RT-induced apoptosis, was significantly enhanced by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activity was associated with a marked increase in Caspase 3/7 activation, indicative of apoptosis. We have previously shown that CONPs increase reactive oxygen species (ROS) production in cancer cells. ROS has been shown to drive the oxidation of thioredoxin 1 (TRX1) which results in the activation of apoptosis signaling kinase 1 (ASK1). The increase in ASK1 activation following the co-treatment with CONPs followed by RT suggests that the increased JNK activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue or when the JNK activation was blocked by an inhibitor. Taken together, these data demonstrate an important mechanism for CONPs in specifically killing cancer cells and provide novel insights into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.

ILDR2 Alters Hepatic Phospholipid Composition via MBOAT7—Effects on Liver Fat

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and increases the risk for liver cirrhosis and cancer. A diabetes susceptibility gene, ILDR2, encodes a transmembrane protein localized to the ER membrane whose primary physiological functions are unclear. We previously purified ILDR2-containing protein complexes using the tandem affinity purification tags and identified MBOAT7, which mediates acyl-chain remodeling of phosphatidylinositols (PIs). The MBOAT7 variant rs641738 T allele is associated with decreased hepatic PI (36:4) and PI (38:3) and an increase in severity of NAFLD. We hypothesized that ILDR2 may influence hepatic Pl composition in NAFLD. We used two mouse models fed a methionine- and choline-deficient (MCD) diet: 1. liver-specific ILDR2 knockout (LKO-ILDR2) using an AAV-Cre/loxP system; and 2. AAV-overexpressing ILDR2 (OX-ILDR2) mice. In LKO-ILDR2 mice compared to AAV-GFP injected ILDR2 floxed (GFP-flox) mice, liver triglyceride (TG) content was significantly increased by 1.6-fold with a significant 1.2-fold increase in plasma TG. In LKO-ILDR2 mice compared to GFP-flox mice, hepatic PI (38:4) was significantly decreased. The livers of LKO-ILDR2 mice showed significantly increased JNK activation, collagen gene expression, and increased in hydroxyproline, suggesting that ILDR2 depletion leads to inflammation and the development of fibrosis. In OX-ILDR2 mice compared to AAV-GFP injected WT (GFP-WT) mice, hepatic PI and PC species were significantly increased. The liver in OX-ILDR2 mice showed decreased expression of JNK and ER stress-related genes, and hydroxyproline, with a slight (9%) decrease in hepatic TG compared to GFP-WT mice. Thus, ILDR2 overexpression attenuated hepatic inflammation, ER stress, and fibrogenesis. Manipulation of hepatic ILDR2 expression is associated with changes in PI and PC molecular species in the liver. Patterns of hepatic phosphatidyl fatty acid composition are associated with NAFLD development. Disclosure K. Watanabe: None. K. Nakayama: None. K. Yoshida: None. E.J. Millings: None. C.A. LeDuc: None. R. Leibel: None. S. Iwamoto: None.