Activation Of Downstream Transcription Factors Research Articles

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signalling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favours geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants do not display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect. This article is protected by copyright. All rights reserved.

A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans

Abstract A20 is an NF-κB-induced, dual function ubiquitin editing enzyme that negatively regulates NF-κB signaling induced by TNF and other innate immune receptors, constraining inflammation. Indeed, A20 haploinsufficiency drives a severe autoinflammatory disease in humans. Although A20 serves a similar negative feedback function for T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun. We employed an extensive set of mutant expression constructs to better elucidate how A20 regulates TCR signaling output in Jurkat T cells. Intriguingly, A20 knockout cells showed enhanced, sustained NF-κB and JNK signaling after stimulation, which was reversed upon wild-type (WT) A20 complementation. A20 deubiquitinase function was entirely dispensable for NF-κB and JNK regulation, whereas zinc finger domains that confer E3 ligase activity (ZnF4) and linear ubiquitin-binding capacity (ZnF7) were required. Abolishing MALT1-dependent proteolytic cleavage of A20 did not affect suppressive function, although neither N- nor C-terminal A20 cleavage fragments retained regulatory capacity. We also found that CRISPR-mediated deletion of A20 adversely affects the survival and expansion of primary human T cells. Ongoing studies will determine how A20 deletion affects human T cell effector responses after stimulation, and decipher how dysregulated NF-κB and/or JNK signaling contributes to T cell-intrinsic abnormalities that perturb adaptive immune homeostasis.

Regulation of floral senescence in Arabidopsis by coordinated action of CONSTANS and jasmonate signaling

In Arabidopsis, photoperiodic flowering is controlled by the regulatory hub gene CONSTANS (CO), whereas floral organ senescence is regulated by the jasmonates (JAs). Because these processes are chronologically ordered, it remains unknown whether there are common regulators of both processes. In this study, we discovered that CO protein accumulates in Arabidopsis flowers after floral induction, and it displays a diurnal pattern in floral organs different from that in the leaves. We observed that altered CO expression could affect flower senescence and abscission by interfering with JA response, as shown by petal-specific transcriptomic analysis as well as CO overexpression in JA synthesis and signaling mutants. We found that CO has a ZIM (ZINC-FINGER INFLORESCENCE MERISTEM) like domain that mediates its interaction with the JA response repressor JAZ3 (jasmonate ZIM-domain 3). Their interaction inhibits the repressor activity of JAZ3, resulting in activation of downstream transcription factors involved in promoting flower senescence. Furthermore, we showed that CO, JAZ3, and the E3 ubiquitin ligase COI1 (Coronatine Insensitive 1) could form a protein complex in planta, which promotes the degradation of both CO and JAZ3 in the presence of JAs. Taken together, our results indicate that CO, a key regulator of photoperiodic flowering, is also involved in promoting flower senescence and abscission by augmenting JA signaling and response. We propose that coordinated recruitment of photoperiodic and JA signaling pathways could be an efficient way for plants to chronologically order floral processes and ensure the success of offspring production.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Epstein-Barr Virus-Encoded BILF1 Orthologues From Porcine Lymphotropic Herpesviruses Display Common Molecular Functionality

Infection of immunosuppressed transplant patients with the human γ-herpesvirus Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD), an often fatal complication. Immunosuppressed miniature pigs infected with γ-herpesvirus porcine lymphotropic herpesvirus 1 (PLHV1) develop a similar disease, identifying pigs as a potential preclinical model for PTLD in humans. BILF1 is a G protein-coupled receptor (GPCR) encoded by EBV with constitutive activity linked to tumorigenesis and immunoevasive function downregulating MHC-I. In the present study, we compared BILF1-orthologues encoded by the three known PLHVs (PLHV1-3) with EBV-BILF1 to determine pharmacological suitability of BILF1 orthologues as model system to study EBV-BILF1 druggability. Cell surface localization, constitutive internalization, and MHC-I downregulation as well as membrane proximal constitutive Gαi signaling patterns were conserved across all BILFs. Only subtle differences between the individual BILFs were observed in downstream transcription factor activation. Using Illumina sequencing, PLHV1 was observed in lymphatic tissue from PTLD-diseased, but not non-diseased pigs. Importantly, these tissues showed enhanced expression of PLHV1-BILF1 supporting its involvement in PTLD infection.

Role of NFAT in Chronic Lymphocytic Leukemia and Other B-Cell Malignancies.

In recent years significant progress has been made in the clinical management of chronic lymphocytic leukemia (CLL) as well as other B-cell malignancies; targeting proximal B-cell receptor signaling molecules such as Bruton Tyrosine Kinase (BTK) and Phosphoinositide 3-kinase (PI3Kδ) has emerged as a successful treatment strategy. Unfortunately, a proportion of patients are still not cured with available therapeutic options, thus efforts devoted to studying and identifying new potential druggable targets are warranted. B-cell receptor stimulation triggers a complex cascade of signaling events that eventually drives the activation of downstream transcription factors including Nuclear Factor of Activated T cells (NFAT). In this review, we summarize the literature on the expression and function of NFAT family members in CLL where NFAT is not only overexpressed but also constitutively activated; NFAT controls B-cell anergy and targeting this molecule using specific inhibitors impacts on CLL cell viability. Next, we extend our analysis on other mature B-cell lymphomas where a distinct pattern of expression and activation of NFAT is reported. We discuss the therapeutic potential of strategies aimed at targeting NFAT in B-cell malignancies not overlooking the fact that NFAT may play additional roles regulating the inflammatory microenvironment.

Fludarabine modulates expression of type VII collagen during haematopoietic stem cell transplantation for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, complicated inherited blistering skin disease with few treatment options currently available. Recently, haematopoietic stem cell transplantation (HCT) has been used as an alternative therapy that can improve skin integrity, but it is not known if the preparative HCT regimen also contributes to the therapeutic response. To determine whether chemotherapy drugs used in the HCT preparative regimen influence type VII collagen (C7) expression, which is inherently reduced or absent in RDEB skin, and to explore the pathomechanisms of such responses, if present. Drugs from the HCT preparative regimen (busulfan, cyclophosphamide, ciclosporin A, fludarabine and mycophenolate) with inhibitors (PD98059, U0126, LY294002, SR11302, SIS3 and N-acetyl-l-cysteine) were added to normal human dermal and human RDEB fibroblasts. C7 expression was measured using reversetranscription polymerase chain reaction and immunoblotting. We uncovered a previously unknown consequence of fludarabine whereby dermal fibroblasts exposed to fludarabine upregulate C7. This effect is mediated, in part, through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B and transforming growth factor-β pathways. Activation of these pathways leads to activation of downstream transcription factors, including activator protein 1 (AP-1) and SMAD. Subsequently, both AP-1 and SMAD bind the COL7A1 promoter and increase COL7A1 expression. Fludarabine influences the production of type VII collagen in RDEB fibroblasts.

Desensitization of ABA-Signaling: The Swing From Activation to Degradation.

Abscisic acid (ABA) is a key plant stress-signaling hormone that accumulates upon osmotic stresses such as drought and high salinity. Several proteins have been identified that constitute the ABA-signaling pathway. Among them ABA receptors (PYR/PYL/RCAR), co-receptor PP2Cs (protein phosphatases), SnRK2 kinases (SNF1-related protein kinases) and ABI5/ABFs (transcription factors) are the major components. Upon ABA signal, PYR/PYL receptors interact with and recruit PP2Cs, releasing SnRK2s kinases from sequestration with PP2Cs. This allows SnKR2s to promote the activation of downstream transcription factors of ABA pathway. However, apart from activation, ubiquitination and degradation of core proteins in the ABA pathway by the ubiquitin proteasome system is less explored. In this review we will focus on the recent findings about feedback regulation of ABA signaling core proteins through degradation, which is emerging as a critical step that modulates and eventually ceases the signal relay. Additionally, we also discuss the importance of the recently identified effector protein HOS15, which negatively regulate ABA-signaling through degradation of OST1.

JCPyV-Induced MAPK Signaling Activates Transcription Factors during Infection.

JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.

Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK.

Clustering of the B cell antigen receptor (BCR) by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of signals sent by the crosslinked BCR, we developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, we fused SYK with E. coli dihydrofolate reductase (eDHFR), which binds the small molecule trimethoprim (TMP) with high affinity and selectivity and synthesized a dimer of TMP with a flexible linker. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of Nuclear Factor of Activated T cells (NFAT) without affecting the activation of NFκB. The dimerization of SYK enhances the duration but not the amplitude of calcium mobilization by enhancing the extent and duration of its interaction with the crosslinked BCR at the plasma membrane.

Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-Derived 3D Pancreatic Cancer Models.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α-mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1-induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment. Mol Cancer Ther; 15(11); 2722-32. ©2016 AACR.

Enzyme-Treated Asparagus Extract Attenuates Hydrogen Peroxide-Induced Matrix Metalloproteinase-9 Expression in Murine Skin Fibroblast L929 Cells

Enzyme-treated asparagus extract (ETAS) exerts a wide variety of beneficial biological actions including facilitating anti-cortisol stress and neurological anti-aging responses. However, the anti-skin aging effects of ETAS remain to be elucidated. Reactive oxygen species (ROS) play pivotal roles in skin aging. Increased ROS levels in fibroblasts in response to ultraviolet irradiation activate c-Jun N-terminal kinase (JNK) and its downstream transcription factor activator protein-1 (AP-1), and the resultant gene expression of matrix metalloproteinase (MMP) isoforms accelerates collagen breakdown in the dermis. Therefore, we explored whether ETAS has anti-skin aging effects by attenuating the oxidative stress responses in fibroblasts. Simultaneous treatment of murine skin L929 fibroblasts with hydrogen peroxide (H2O2) and either ETAS or dextrin showed that ETAS significantly suppressed H2O2-induced expression of MMP-9 mRNA as measured by real-time polymerase chain reaction. ETAS also clearly suppressed H2O2-stimulated phosphorylation of c-Jun (AP-1 subunit) and JNK as determined by Western blot. However, ETAS did not affect the increased amounts of carbonyl proteins in response to H2O2, also as determined by Western blotting. These results suggest that ETAS diminishes cellular responsiveness to ROS but does not scavenge ROS. Thus, ETAS has the potential to prevent skin aging through attenuating the oxidative stress responses in dermal fibroblasts.

Interleukin‐6 Transactivation of the Mineralocorticoid Receptor

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone‐sensitive distal nephron (ASDN) as well as a chronic systemic inflammation. Interleukin‐6 (IL‐6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone which modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. We hypothesize that IL‐6 can transactivate the MR, increasing Na+ reabsorption via reactive oxygen species (ROS) generation and activation of the sodium‐chloride cotransporter (NCC). We transfected MR‐EGFP tagged constructs into a model of murine distal convoluted tubule (mDCT15) cells. Cells were then treated with vehicle, Aldo (100–250nM) or IL‐6 (100ng/mL), fixed and visualized with confocal microscopy. MR‐EGFP expression was observed in the cytosol in the vehicle treated cells. However, following IL‐6 treatment (30 min), MR‐EGFP fluorescence was observed translocated into the nucleus, similar to Aldo‐treated positive controls. To confirm activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)‐luciferase reporter constructs, treated with vehicle, Aldo (100nM, 24hr) or IL‐6 (100ng/mL, 24hr) and luciferase assays performed (48hrs). With IL‐6 treatment, luciferase activity was increased approximately 1.5 fold over MRE‐only transfected cells. Excessive MR stimulation is known to produce ROS generation. Thus, we determined if ROS generation stimulates increased NCC‐mediated Na+ uptake. Using mDCT15 cell monolayers, thiazide‐sensitive 22Na+‐uptake studies were performed. Increased 22Na+ uptake was observed after ROS generation (pyrogallol, 50μM) as compared to vehicle (1834±19 vs. 1506±18nmol/mg, p<0.0001, n=6), which was then reduced with the addition of the superoxide dismutase mimetic tempol (pyrogallol+tempol, 250μM) (1618±27nmol/mg vs. pyrogallol+tempol, p<0.0001, n=6). These data suggest that: IL‐6 can transactivate the MR leading to nuclear translocation and subsequent MRE activation, and ROS generation increases ASDN Na+ reabsorption via NCC providing evidence for alternate mechanisms of ASDN Na+ uptake during conditions where Aldo‐mediated MR stimulation may not occur.Support or Funding InformationNIDDK085097‐RSH2T32DK7656‐21 Emory Renal Division‐BMW

Temporal Modulation of a GPCR Pathway Elucidates Band-Pass Processing for the Downstream Signaling and Transcription Factor Activation

G-protein coupled receptors (GPCRs) are major pharmaceutical targets. Quantitative understanding of GPCR signaling pathway architecture has been limited by the temporal observability space available in the dose-response studies. We use a microfluidic platform that delivers temporally modulated step and pulsatile ligands to single HEK293 cells with simultaneous real-time assessment of downstream calcium signal and calcium-regulated transcription factor activation. We pair these experiments with computational modeling to delineate the mechanism of frequency and amplitude modulation by GPCRs. We find that the pulsatile ligand input, when timed appropriately, leads to better recovery of the calcium signal as well as greater downstream transcription factor activation as compared to step changes in the ligand concentration. Further, we also show that receptor kinetics and the dynamics of downstream signaling combine to form a band-pass regime of frequencies for which the signaling is significantly enhanced. Computational modeling suggests that the band-pass nature of signaling pathways may be a generic theme wherein an upstream module acts as a low-pass filter to distinguish signal from noise, and the downstream module acts a high pass filter to amplify signals received. We further delineate the low pass filter characteristics of the GPCR in single cells and show that cell-to-cell variability may lead to distinguishable downstream responses that may not be obvious by looking at the population scale response. These findings may facilitate the design of therapeutic interventions and the development of enhanced in vitro cell culture protocols.

MNG promotes neurogenesis against cerebral ischemic injury (LB626)

MNG is an active component of sarcocarp of C. officinalis that have shown neuroprotective effects. The aim of the present study is to test whether MNG promote neurogenesis via Wnt/β‐catenin signaling pathway for brain recovery in a rat model of focal cerebral ischemia. MNG were administered intragastrically once daily at the concentrations of 30, 90 and 270 mg/kg respectively for 7 days post‐ischemia. Ludmila Belayev score tests was performed to detect neurological function. Immunofluorescence staining of Nestin was used to identify the endogenous neural stem cells proliferation in the subventricular zone. The expression of proteins involved in and related to Wnt/β‐catenin signaling pathway was detected by western blotting analysis. MNG significantly promoted neurogenesis for brain recovery 7 days post ischemia. Increased expression of Wnt 3a and β‐catenin, along with activation of downstream transcription factors, indicated that the neurorestorative effects of MNG may be associated with Wnt/β‐catenin signaling pathway. These data provide support for understanding the mechanisms of MNG in neurorestorative effects and suggest a potential new strategy for stroke treatment.Grant Funding Source: Supported by the National Science and Technology Major Project 2012ZX09102201‐106.

Finding a fairy in the forest: ELF4, a novel and critical element of type I interferon responses

Type I interferon (IFN) production is a key event during innate immune responses. This early and prompt mechanism is mediated by multiple factors that involve various pattern recognition receptors (PRRs), adaptor proteins, kinases and transcription factors. Stimulator of IFN genes (STING) is an important transmembrane adaptor protein that plays a role in the activation of downstream transcription factors, such as IFN regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6, via TANK-binding kinase 1 (TBK1), which has been considered the main innate immune defense weaponry against viruses and intracellular pathogens. In a current issue of Nature Immunology, You et al. showed that, after viral infection, the ETS-related transcription factor ELF4 (previously known as MEF) interacts with STING and is consequently activated by TBK1. This process leads to the nuclear translocation of ELF4 and its binding to IFN promoters (Figure 1). The group also demonstrated that ELF4 acts as an IFN transcription factor, as it has the potential to increase the binding affinity of IRF3 and IRF7 through cooperative binding to newly identified enhancer elements (EICE) in the IFN promoters.1 These new findings not only extend our knowledge about the details of the fine-tuning of the innate IFN responses, but also represent a new milestone by describing the role of EICE elements in this process.

Oxidative Stress–Induced JNK1/2 Activation Triggers Proapoptotic Signaling and Apoptosis That Leads to Diabetic Embryopathy

Oxidative stress and apoptosis are implicated in the pathogenesis of diabetic embryopathy. The proapoptotic c-Jun NH2-terminal kinases (JNK)1/2 activation is associated with diabetic embryopathy. We sought to determine whether 1) hyperglycemia-induced oxidative stress is responsible for the activation of JNK1/2 signaling, 2) JNK1 contributes to the teratogenicity of hyperglycemia, and 3) both JNK1 and JNK2 activation cause activation of downstream transcription factors, caspase activation, and apoptosis, resulting in neural tube defects (NTDs). Wild-type (WT) embryos from nondiabetic WT dams and WT, superoxide dismutase (SOD)1–overexpressing, jnk1+/−, jnk1−/−, and jnk2−/− embryos exposed to maternal hyperglycemia were used to assess JNK1/2 activation, NTDs, activation of transcription factors downstream of JNK1/2, caspase cascade, and apoptosis. SOD1 overexpression abolished diabetes-induced activation of JNK1/2 and their downstream effectors: phosphorylation of c-Jun, activating transcription factor 2, and E twenty-six–like transcription factor 1 and dephosphorylation of forkhead box class O3a. jnk1−/− embryos had significantly lower incidences of NTDs than those of WT or jnk1+/− embryos. Either jnk1 or jnk2 gene deletion blocked diabetes-induced activation of JNK1/2 signaling, caspases 3 and 8, and apoptosis in Sox1+ neural progenitors of the developing neural tube. Our results show that JNK1 and JNK2 are equally involved in diabetic embryopathy and that the oxidative stress–JNK1/2–caspase pathway mediates the proapoptotic signals and the teratogenicity of maternal diabetes.

Instructive cytokine signals in dendritic cell lineage commitment

Clarifying the signals that lead to dendritic cell (DC) development and identifying cellular intermediates on their way to DC differentiation are essential steps to understand the dynamic regulation of number, localization, and functionality of these cells. In the past decade, much knowledge on cytokines, transcription factors, and successive progenitors involved in steady-state and demand-adapted DC development was gained. From the stage of multipotent progenitors, DCs are generated from Flt3(+) intermediates, irrespective of lymphoid or myeloid commitment, making fms-related tyrosine kinase 3 ligand one of the major regulators for DC development. Additional key cytokines involved are granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, with each being essential for particular DC subsets and leading to specific activation of downstream transcription factors. In this review, we seek to draw an integrative view on how instructive cytokine signals acting on intermediate progenitors might lead to the generation of specific DC subsets in steady-state and during inflammation. We hypothesize that the lineage potential of a progenitor might be determined by the set of cytokine receptors expressed that make it responsive to further receive lineage instructive signals. Commitment to a certain lineage might consequently occur when lineage-relevant cytokine receptors are further upregulated and others for alternative lineages are lost. Along this line, we emphasize the role that diverse microenvironments have in influencing the generation of DC subsets with specific functions throughout the body.

Induction of MAPK-dependent transcription factors by deoxynivalenol in human cell lines

Contamination of cereals with the trichothecene deoxynivalenol (DON) is a global problem in agriculture. DON-related cytotoxicity results from inhibition of translation preceded by a ribotoxic stress response, which is characterized by phosphorylation of the mitogen-activated protein kinases and activation of downstream transcription factors. In this study, we measured the expression of AP-1 associated transcription factor mRNA levels in six human cell lines (Hep-G2, A549, U937, A204, Jurkat, and CaCo-2) by using real-time RT-PCR. Previous work suggested that transcription factors mRNA and protein levels are affected by DON in Hep-G2 cells. In this study, we found significant cell-specific differences in mRNA levels for the transcription factors JUN, JUND, FOS, FOSL2, ATF3, and EGR1. After exposure to 1µmol/l DON for 3h, the induction of the transcription factor JUN was highest in the Jurkat (342-fold) and Hep-G2 (84-fold) cell lines. JUND expression was mainly affected in the immunocompetent cell-lines U937 (11-fold) and Jurkat (12-fold), whereas significant FOSL2 induction (5-fold) was only found in Jurkat cells. DON-induced expression of FOS was mainly observed in Hep-G2 (96-fold) and U937 cells (59-fold). In contrast, response of A549 cells to DON was characterized by a distinct induction of ATF3 (44-fold) and EGR1 (92-fold). Time- and concentration-dependent induction of the transcription factors by DON was studied in detail for Hep-G2, A549, A204, and U937 cells. Considering the chronic dietary exposure of humans, broader investigations on DON influence on cell signaling pathways are required to understand the impact of this mycotoxin on human health.

Differential induction of innate anti-viral responses by TLR ligands against Herpes simplex virus, type 2, infection in primary genital epithelium of women

Genital epithelial cells (GECs) are the first line of mucosal defense against sexually transmitted infections. We exploited the ability of GECs to mount innate immune responses, by using TLR ligands to induce anti-viral activity against Herpes simplex virus, type 2 (HSV-2). Primary cultures of GECs were grown to confluent, polarized monolayers and found to express different levels of mRNA for TLR1-10. Innate anti-viral responses against HSV-2 infection were determined following treatment with eight different TLR ligands. HSV-2 replication was significantly inhibited following treatment with ligands for TLR3, 5 and 9, while lipo-polysaccharide (LPS), a TLR4 ligand, failed to provide any protection. Biologically active interferon-beta and nitric oxide production by GECs correlated with anti-viral activity. Following treatment with TLR3 ligand Poly I:C, inflammatory cytokines were upregulated. Poly I:C treatment led to activation of downstream transcription factors including interferon regulatory factor-3 (IRF-3) and NFkappaB. Anti-viral responses induced by TLR ligands in GECs may provide a unique alternative to topical microbicides by enhancing body's own mucosal innate defense mechanisms against sexually transmitted viruses.