BACKGROUND:Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a key regulator of signal cascades of TNF-α receptor and TLR4, and can induce NF-κB activation for preventing cell apoptosis and eliciting inflammation response.RESULTS:TAK1 inhibitor (TAKI) can decrease the cell viability of murine bone marrow-derived macrophages (BMDM), RAW264.7 and BV-2 cells, but not dermal microvascular endothelial cells, normal human epidermal keratinocytes, THP-1 monocytes, human retinal pigment epithelial cells, microglia CHME3 cells, and some cancer cell lines (CL1.0, HeLa and HCT116). In BMDM, TAKI-induced caspase activation and cell apoptosis were enhanced by lipopolysaccharide (LPS). Moreover, TAKI treatment increased the cytosolic and mitochondrial reactive oxygen species (ROS) production, and ROS scavengers NAC and BHA can inhibit cell death caused by TAKI. In addition, RIP1 inhibitor (necrostatin-1) can protect cells against TAKI-induced mitochondrial ROS production and cell apoptosis. We also observed the mitochondrial membrane potential loss after TAKI treatment and deterioration of oxygen consumption upon combination with LPS. Notably TNF-α neutralization antibody and inhibitor enbrel can decrease the cell death caused by TAKI.CONCLUSIONS:TAKI-induced cytotoxicity is cell context specific, and apoptosis observed in macrophages is dependent on the constitutive autocrine action of TNF-α for RIP1 activation and ROS production.

NLRP3 is the most crucial member of the NLR family, as it detects the existence of pathogen invasion and self-derived molecules associated with cellular damage. Several studies have reported that excessive NLRP3 inflammasome-mediated caspase-1 activation is a key factor in the development of diseases. Recent studies have reported that Syk is involved in pathogen-induced NLRP3 inflammasome activation; however, the detailed mechanism linking Syk to NLRP3 inflammasome remains unclear. In this study, we showed that Syk mediates NLRP3 stimuli-induced processing of procaspase-1 and the consequent activation of caspase-1. Moreover, the kinase activity of Syk is required to potentiate caspase-1 activation in a reconstituted NLRP3 inflammasome system in HEK293T cells. The adaptor protein ASC bridges NLRP3 with the effector protein caspase-1. Herein, we find that Syk can associate directly with ASC and NLRP3 by its kinase domain but interact indirectly with procaspase-1. Syk can phosphorylate ASC at Y146 and Y187 residues, and the phosphorylation of both residues is critical to enhance ASC oligomerization and the recruitment of procaspase-1. Together, our results reveal a new molecular pathway through which Syk promotes NLRP3 inflammasome formation, resulting from the phosphorylation of ASC. Thus, the control of Syk activity might be effective to modulate NLRP3 inflammasome activation and treat NLRP3-related immune diseases.

The immediate early gene c-Fos is reported to be regulated by Elk-1 and cAMP response element-binding protein (CREB), but whether nuclear factor (NF)-κB is also required for controlling c-Fos expression is unclear. In this study, we determined how NF-κB’s coordination with Elk/serum response factor (SRF) regulates c-fos transcription. We report that PMA strongly induced c-Fos expression, but tumor necrosis factor (TNF)-α did not. In mouse embryonic fibroblasts, the PMA induction of c-Fos was suppressed by a deficiency in IKKα, IKKβ, IKKγ, or p65. By contrast, in human embryonic kidney 293 cells, PMA induced c-Fos independently of p65. In accordance with these results, we identified an NF-κB binding site in the mouse but not human c-fos promoter. Under PMA stimulation, IKKα/β mediated p65 phosphorylation and the binding of the p65 homodimer to the NF-κB site in the mouse c-fos promoter. Furthermore, our studies demonstrated independent but coordinated functions of the IKKα/β-p65 and extracellular signal-regulated kinase (ERK)-Elk-1 pathways in the PMA induction of c-Fos. Collectively, these results reveal the distinct requirement of NF-κB for mouse and human c-fos regulation. Binding of the p65 homodimer to the κB site was indispensable for mouse c-fos expression, whereas the κB binding site was not present in the human c-fos promoter. Because of an inability to evoke sufficient ERK activation and Elk-1 phosphorylation, TNF-α induces c-Fos more weakly than PMA does in both mouse and human cells.

Extracellular ATP is an important signaling molecule mediating quite divergent specific biological effects. Even though recent studies suggest a potential role of ATP in cancer progress, its real impact in chemotherapeutic efficacy remains unclear. In the present study, we investigated the effect of ATP on the cytotoxicity of doxorubicin in various cancer cell types and found that ATP had no effect on doxorubicin cytotoxicity in colon, prostate, breast, and cervical cancers or in osteosarcoma. In contrast, ATP has divergent effects on lung cancer cells: it can protect against doxorubicin‐induced cell death in non‐metastatic lung cancer CL1.0 cells, but not in highly metastatic CL1.5 cells. Both apoptotic (characterized by sub‐G1 peak, caspase 3 activation, poly(ADP‐ribose) polymerase‐1 cleavage) and necrotic (characterized by propidium iodide uptake and ROS production) features induced by doxorubicin in CL1.0 cells were reduced by ATP. In addition, ATP attenuated p53 accumulation, DNA damage (assessed by poly(ADP‐ribose) formation and the comet assay) and topoisomerase II inhibition after doxorubicin treatment, and doxorubicin cytotoxicity was diminished by the p53 inhibitor pifithrin‐α. Moreover, UTP, UDP, ADP, and pyrophosphate sodium pyrophosphate tetrabasic decahydrate diminished the antitumor effect of doxorubicin in CL1.0 cells, whereas purinergic P2 receptors antagonists did not abrogate the action of ATP. In summary, ATP fails to alter the antitumor efficacy of doxorubicin in most cancer cell types, except in CL1.0 cells, in which pyrophosphate mediates the cell protection afforded by ATP via attenuation of reactive oxygen species production, DNA damage, p53 accumulation, and caspase activation.

3-Methyladenine (3-MA) is one of the most commonly used inhibitors in autophagy research today. However, rather than inhibiting class III PI3K that is involved in autophagy suppression, 3-MA might also interfere with class I PI3K and consequently augment autophagy flux. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in TLR4-mediated inflammatory responses in RAW264.7 macrophages and, moreover, to decipher the action of 3-MA in modulation of autophagy. We found that 3-MA could enhance LPS-induced NF-kappa B activation and production of TNF-alpha, inducible NO synthase (iNOS), cyclooxygenase-2, IL-1 beta, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-beta production and STAT signaling. Studies revealed that 3-MA can, through inhibition of Akt as a result of class I PI3K interference, positively regulate p38, JNK, and p65, but negatively regulate TANK-binding kinase 1 and IFN regulatory factor 3 mediated by TLR4. As glycogen synthase kinase 3 beta (GSK3 beta) is an important Akt substrate, we further explored its involvement in the actions of 3-MA. 3-MA was found to enhance LPS-induced NF-kappa B activation, iNOS, and pro-IL-1 beta expression, and these actions were reversed by either GSK3 beta inhibitors or small interfering GSK3 beta. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macrophages, but the stimulating effects on NF-kappa B activation and iNOS and cyclooxygenase-2 expression were not affected in LPS-stimulated macrophages with small interfering autophagy protein-5 treatment. These results not only shed new light on the action mechanisms of 3-MA to differentially regulate inflammatory outcomes derived from TLR4-mediated MyD88 and Toll/IL-1R domain-containing adapter inducing IFN-beta pathways, but also highlight the necessity to check autophagy status upon taking 3-MA as a general autophagy inhibitor. The Journal of Immunology, 2012, 189: 4154-4164.

The multiple cytoprotective mechanisms of heme oxygenase (HO)-1 make it a promising therapeutic target. This study investigated whether the selective cyclooxygenase (COX)-2 inhibitor, celecoxib, can upregulate HO-1 expression. Murine J774 macrophages and rat aortic vascular smooth muscle cells (VSMCs) were used to study the effect of celecoxib on HO-1 expression. A signal transduction pathway involving reactive oxygen species (ROS) was also investigated. We found that celecoxib can upregulate HO-1 gene and protein expressions in J774 macrophages and VSMCs. This effect was not diminished by prostaglandin E2 or 15dPGJ2, while it was additive to hypoxia-induced HO-1 expression, suggesting an event independent of COX-2 activity or hypoxia-inducible factor-1α. Moreover, celecoxib activated ERK, p38, Akt, and Nrf2 as well as increased ROS production. All these events contributed to the increase in the expression of HO-1 caused by celecoxib. In this study, we also, for the first time, demonstrated that AMP-activated protein kinase (AMPK) can mediate HO-1 expression via the downstream activation of p38 and Akt. However, the HO-1-inducing actions of celecoxib and hypoxia were not associated with AMPK. This study demonstrates a COX-2-independent action of celecoxib in upregulating HO-1 in macrophages and VSMCs. This action is dependent on ROS, Akt, ERK, p38, and Nrf2 activation. These findings provide new insights into the action mechanism of celecoxib with broad implications for anti-inflammation therapy.

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.

AMP-activated protein kinase (AMPK), a critical signaling molecule for regulating energy homeostasis, might bi-directionally regulate inflammation, and its action mechanism leading to inflammation is not fully understood. We utilized 5-aminoimidazole-4-carboxamide riboside (AICAR) as a pharmacological activator of AMPK to unveil the effects of and signaling cascades mediated by AMPK on cyclooxygenase (COX)-2 gene expression in rat aortic vascular smooth muscle cells (VSMCs), murine macrophage cell line (J774), and human umbilical vein endothelial cells (HUVECs). Biochemical approaches were further conducted to elucidate interactions among signaling molecules. We found that AICAR could induce COX-2 protein expression in the cell types tested. This event was mediated by COX-2 gene transcription, and abrogated by compound C and 5'-iodotubercidin, suggesting the essential role of AMPK in COX-2 induction. Pharmacological and biochemical studies indicated that p38 mitogen-activated protein kinase (MAPK) activation is the common downstream signal of AMPK in COX-2 expression in all three cell types. Furthermore, we also found that TAK1 is associated with AMPKalpha2, and this binding requires an interaction between the kinase domains of both molecules. Notably data of TAK1 phosphorylation indicate that the activating state is enhanced upon AMPK activation in vivo and in vitro. Our data for the first time prove a pivotal role of TAK1 in the AMPK signaling axis. Such interaction gives AMPK an additional pathway for regulating cellular functions. Via a downstream p38 MAPK signaling cascade, AMPK-dependent TAK1 activation leads to the expression of the inflammatory COX-2 gene in various cell types.

Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.

Among the 162 Candida tropicalis isolates collected in the Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 1999, 23 (14.2 %) had fluconazole MICs > or = 64 mg l(-1), and thus fulfilled the definition of resistance. Random amplified polymorphic DNA assay showed that all 23 fluconazole-resistance C. tropicalis isolates collected from different hospitals around Taiwan were closely related. Two distinct pulsotypes associated with fluconazole susceptibility were identified when these 23 resistant isolates, along with 13 susceptible ones, were analysed by PFGE.

Background In the past decades, the prevalence of candidemia has increased significantly and drug resistance has also become a pressing problem. Overexpression of CDR1 , an efflux pump, has been proposed as a major mechanism contributing to the drug resistance in Candida albicans . It has been demonstrated that biological fluids such as human serum can have profound effects on antifungal pharmacodynamics. The aim of this study is to understand the effects of serum in drug susceptibility via monitoring the activity of CDR1 promoter of C. albicans . Results The wild-type C. albicans cells (SC5314) but not the cdr1/cdr1 mutant cells became more susceptible to the antifungal drug when the medium contained serum. To understand the regulation of CDR1 in the presence of serum, we have constructed CDR1 promoter-Renilla luciferase ( CDR1p-RLUC ) reporter to monitor the activity of the CDR1 promoter in C. albicans . As expected, the expression of CDR1p-RLUC was induced by miconazole. Surprisingly, it was repressed by serum. Consistently, the level of CDR1 mRNA was also reduced in the presence of serum but not N-acetyl-D-glucosamine, a known inducer for germ tube formation. Conclusion Our finding that the expression of CDR1 is repressed by serum raises the question as to how does CDR1 contribute to the drug resistance in C. albicans causing candidemia. This also suggests that it is important to re-assess the prediction of in vivo therapeutic outcome of candidemia based on the results of standard in vitro antifungal susceptibility testing, conducted in the absence of serum.

CaNdt80p, the Candida albicans homologue of the Saccharomyces cerevisiae transcription factor ScNdt80p, has been identified as a positive regulator of CDR1, which encodes an efflux pump involved in drug resistance in C. albicans. To investigate the involvement of the putative DNA-binding domain of CaNdt80p in drug resistance, chimeras of CaNdt80p and ScNdt80p were constructed. Interestingly, the DNA-binding domain of ScNdt80p could functionally complement that of CaNdt80p to activate CDR1p-lacZ in S. cerevisiae. Consistently, CaNdt80p containing a mutation in the DNA-binding domain failed to activate CDR1p-lacZ in S. cerevisiae. Furthermore, a copy of CaNDT80 with the same mutation also failed to complement the drug-sensitive phenotype caused by a null mutation in C. albicans. Thus, the DNA-binding domain of CaNdt80p is critical for its function in drug resistance in C. albicans.

The aquatic fern Marsilea quadrifolia produces different types of leaves in response to changes in natural environment and culture conditions. When the conditions are in favor of producing the submerged-type leaves, exogenous application of the plant hormone abscisic acid (ABA) induces the formation of aerial-type leaves. Tissues responsive to ABA were localized to the shoot apical meristem and the associated organ primordia. From these tissues, at least two tiers of ABA-regulated early genes were identified, including seven primary genes and seventeen secondary genes. These genes, designated ABRH for ABA-responsive heterophylly, showed diverse expression patterns during the course of heterophyllous induction. Changes in the transcript level of ABRH genes started early, within 0.5–1.0 h after the addition of ABA to the culture medium. Some changes were transient while the others were persistent. The ABRHs contain extensive sequence homology to known genes, including those encoding transcription factors, protein kinases, membrane transporters, metabolic enzymes, structural proteins and those encoded by the chloroplast genome. Identification of these ABRHs is a first step toward the understanding of the regulation mechanisms of heterophylly, and the results suggest the involvement of novel metabolic and regulatory pathways in ABA-controlled morphogenesis.

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.

The aquatic fern produces different types of leaves in response to changes in natural environment and culture conditions. When the conditions are in favor of producing the submerged-type leaves, exogenous application of the plant hormone abscisic acid (ABA) induces the formation of aerial-type leaves. Tissues responsive to ABA were localized to the shoot apical meristem and the associated organ primordia. From these tissues, at least two tiers of ABA-regulated early genes were identified, including seven primary genes and seventeen secondary genes. These genes, designated for ABA-responsive heterophylly, showed diverse expression patterns during the course of heterophyllous induction. Changes in the transcript level of genes started early, within 0.5–1.0 h after the addition of ABA to the culture medium. Some changes were transient while the others were persistent. The s contain extensive sequence homology to known genes, including those encoding transcription factors, protein kinases, membrane transporters, metabolic enzymes, structural proteins and those encoded by the chloroplast genome. Identification of these s is a first step toward the understanding of the regulation mechanisms of heterophylly, and the results suggest the involvement of novel metabolic and regulatory pathways in ABA-controlled morphogenesis.