The role of NF-kappaB in the angiogenic response of coronary microvessel endothelial cells.

The activation of nuclear factor (NF)-kappaB by 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], an arachidonic acid metabolite with potent stereospecific proinflammatory and angiogenic properties, was examined and its role in the angiogenic response was determined in capillary endothelial cells derived from coronary microvessels. Electrophoretic mobility-shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE demonstrated a rapid and stereospecific time- and concentration-dependent increase in the binding activity of NF-kappaB, which was inhibitable by the antioxidants N-acetylcysteine, butylated hydroxyanisole, and pyrrolidine dithiocarbamate and was partially attenuated by the protein kinase C inhibitors, staurosporine and calphostin C. Neither 12(S)-HETrE nor other related eicosanoids--e.g., 12(R)-HETE, 12(S)-HETE, and leukotriene B4--stimulated the activation of NF-kappaB relative to 12(R)-HETrE, substantiating the claim for a specific receptor-mediated mechanism. 12(R)-HETrE stimulated the formation of capillary-like cords of microvessel endothelial cells distinguishable from a control; this effect was comparable to that observed with basic fibroblast growth factor (bFGF). Inhibition of NF-kappaB activation resulted in inhibition of capillary-like formation of endothelial cells treated with 12(R)-HETrE by 80% but did not affect growth observed with bFGF. It is suggested that 12(R)-HETrE's angiogenic activity involves the activation of NF-kappaB, possibly via protein kinase C stimulation and the generation of reactive oxygen intermediates for downstream signaling.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

Characterization of the promoter region of the mouse Xist gene.

The mouse Xist gene is expressed exclusively from the inactive X chromosome and may be implicated in initiating X inactivation. To better understand the mechanisms underlying the control of Xist expression, we investigated the upstream regulatory region of the mouse Xist promoter. A 1.2-kb upstream region of the Xist gene was sequenced and promoter activity was studied by chloramphenicol acetyltransferase (CAT) assays after transfection in murine XX and XY cell lines. The region analyzed (-1157 to +917 showed no in vitro sex-specific promoter activity. However, a minimal constitutional promoter was assigned to a region from -81 to +1, and a cis element from -41 to -15 regulates promoter activity. We showed that a nuclear factor binds to an element located at -30 to -25 (TTAAAG). A second sequence at -41 to -15 does not act as an enhancer and is unable to confer transcriptional activity to the Xist gene on its own. A third region from -82 to -41 is needed for correct expression. Deletion of the segment -441 to -231 is associated with an increase in CAT activity and may represent a silencer element.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

Isolation of a differentially regulated splicing isoform of human NF-E2.

The transcription factor NF-E2 (nuclear factor erythroid 2), interacting via DNA motifs within regulatory regions of several hematopoietic genes, is thought to mediate the enhancer activity of the globin locus control regions. By screening a human fetal liver cDNA library with probes derived from mouse NF-E2, we have isolated a splicing variant of the NF-E2 gene (fNF-E2) that differs in the 5' untranslated region from the previously reported cDNA (aNF-E2). The fNF-E2 isoform is transcribed from an alternative promoter located in the 3' end of the first intron and joined by alternative splicing to the second and third exons, which are shared by both RNA isoforms. Although the two forms produce the same protein, they are expressed in different ratios during development. fNF-E2 is more abundant in the fetal liver and less abundant in the adult bone marrow compared to the previously described form. Their distribution apparently follows the differential expression of fetal and adult hemoglobins.

Inhibition of NF-kB activation and cytokines production in THP-1 monocytes by 2-styrylchromones

Nuclear factor kappa B (NF-kB) is one of the most important transcription factors whose modulation triggers a cascade of signaling events, namely the expression of many cytokines, enzymes, chemokines, and adhesion molecules, some of which being potential key targets for intervention in the treatment of inflammatory conditions. The 2-styrylchromones (2-SC) designation represents a well-recognized group of natural and synthetic chromones, vinylogues of flavones (2-phenylchromones). Several 2-SC were recently tested for their anti-inflammatory potential, regarding the arachidonic acid metabolic cascade, showing some motivating results. In addition, several flavones with structural similarities to 2-SC have shown NF-kB inhibitory properties. Hence, the aim of the present work was to continue the investigation on the interference of 2-SC in inflammatory pathways. Herein we report their effects on lipopolysaccharide (LPS)-induced NF-kB activation and consequent production of proinflammatory cytokines/chemokine, using a human monocytic cell line (THP-1). From the twelve 2-SC tested, three of them were able to significantly inhibit the NF-kB activation and to reduce the production of the proinflammatory cytokines/chemokine. The compound 3',4',5-trihydroxy-2- styrylchromone stood up as the most active in both assays, being a promising candidate for an anti-inflammatory drug.

Pioglitazone increases renal tubular cell albumin uptake but limits proinflammatory and fibrotic responses

Background. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARgamma agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells. Methods. Cells were exposed to pioglitazone (10 mumol/L) in the presence and absence of low-density lipoprotein (LDL) 100 mug/mL +/- exposure to albumin 1 mg/mL. Results were expressed relative to control (5 mmol/L glucose) conditions. Results. Pioglitazone caused a dose-dependent increase in tubular cell albumin uptake (P < 0.0001). Despite the increase in albumin reabsorption, no concurrent increase in inflammatory or profibrotic markers were observed. Exposure to LDL increased monocyte chemoattractant protein-1 (MCP-1) (P < 0.05) and transforming growth factor-beta1 (TGF-beta1) (P < 0.05) production. which were reversed in the presence of pioglitazone. LDL induced increases in MCP-1 and TGF-β1 were independent of nuclear factor-κB (NF-κB) transcriptional activity. In contrast. tubular exposure to albumin increased tubular protein uptake, in parallel with an increase in MCP-1 (P = 0.05): TGF-β1 (P < 0.02) and NF-kappaB transcriptional activity (P < 0.05). which were unaffected by concurrent exposure to pioglitazone. Conclusion. These findings suggest that dyslipidemia potentiates renal pathology through mechanisms that may be modified PPARγ activation independent of NF-κB transcriptional activitv. In contrast, tubular exposure to protein induces renal damage through NF-κB-dependent mechanisms that are Unaffected by PPARγ activation.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Mechanisms of axon guidance in the developing nervous system

The human brain assembles an incredible network of over a billion neurons. Understanding how these connections form during development in order for the brain to function properly is a fundamental question in biology. Much of this wiring takes place during embryonic development. Neurons are generated in the ventricular zone, migrate out, and begin to differentiate. However, neurons are often born in locations some distance from the target cells with which they will ultimately form connections. To form connections, neurons project long axons tipped with a specialized sensing device called a growth cone. The growing axons interact directly with molecules within the environment through which they grow. In order to find their targets, axonal growth cones use guidance molecules that can either attract or repel them. Understanding what these guidance cues are, where they are expressed, and how the growth cone is able to transduce their signal in a directionally specific manner is essential to understanding how the functional brain is constructed. In this chapter, we review what is known about the mechanisms involved in axonal guidance. We discuss how the growth cone is able to sense and respond to its environment and how it is guided by pioneering cells and axons. As examples, we discuss current models for the development of the spinal cord, the cerebral cortex, and the visual and olfactory systems. (c) 2005, Elsevier Inc.

Does the p75 neurotrophin receptor mediate A beta-induced toxicity in Alzheimer's disease?

Alzheimer's disease is characterized by the over-production and accumulation of amyloidogenic A beta peptide, which can induce cell death in vitro. It has been suggested that the death signal could be transduced by the pan neurotrophin receptor (p75NTR). p75NTR is well known for its ability to mediate neuronal death in neurodegenerative conditions and is inextricably linked with changes that occur in Alzheimer's disease. Moreover, A beta binds to p75NTR, activating signalling cascades. However, the complexity of p75NTR-mediated signalling, which does not always promote cell death, leaves open the possibly of A beta promoting death via an alternative signalling pathway or the regulation of other p75NTR-mediated actions. This review focuses on the interactions between A beta and p75NTR in the context of the broader p75NTR signalling field, and offers alternative explanations for how p75NTR might contribute to the aetiology of Alzheimer's disease.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway

The potential role of 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) as an intracellular signal for increased protein catabolism and induction of the expression of key components of the ubiquitin-proteasome proteolytic pathway induced by a tumour cachectic factor, proteolysis-inducing factor has been studied in murine C2C12 myotubes. 15(S)-HETE induced protein degradation in these cells with a maximal effect at concentrations between 78 and 312 nM. The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There was an increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome, in the same concentration range as that inducing total protein degradation, and this effect was also attenuated by EPA. 15(S)-hydroxyeicosatetraenoic acid also increased maximal expression of mRNA for proteasome subunits C2 and C5, as well as the ubiquitin-conjugating enzyme, E214k, after 4 h incubation, as determined by quantitative competitive RT-PCR. The concentrations of 15-HETE affecting gene expression were the same as those inducing protein degradation. Western blotting of cellular supernatants of myotubes treated with 15(S)-HETE for 24 h showed increased expression of p42, an ATPase subunit of the regulatory complex at similar concentrations, as well as a decrease in expression of myosin in the same concentration range. 15(S)-hydroxyeicosatetraenoic acid activated binding of nuclear factor-κB (NF-κB) in the myotube nucleus and stimulated degradation of 1-κBα. The effect on the NF-κB/1-κBα system was attenuated by EPA. In addition, the NF-κB inhibitor peptide SN50 attenuated the increased chymotrypsin-like enzyme activity in the presence of 15(S)-HETE. These results suggest that 15(S)-HETE induces degradation of myofibrillar proteins in differentiated myotubes through an induction of an increased expression of the regulatory components of the ubiquitin-proteasome proteolytic pathway possibly through the intervention of the nuclear transcription factor NF-κB, and that this process is inhibited by EPA. © 2003 Cancer Research UK.

Biochemical mechanisms of cellular catabolism

Purpose of review: To provide an in-depth analysis of current developments concerning biochemical mechanisms of cellular catabolism. There have been a number of important developments in this area over the past 12 months, particularly with respect to protein catabolism. Recent findings: Protein degradation in a range of catabolic conditions is mediated primarily through the ubiquitin-proteasome proteolytic pathway. Glucocorticoids have been suggested to activate this system in sepsis, while in cancer cachexia a tumour-produced sulphated glycoprotein, proteolysis-inducing factor, induces protein catabolism in skeletal muscle by increasing expression of proteasome subunits and the ubiquitin carrier protein, E214k. Apoptosis may also be important in the loss of muscle protein during the early stage of cachexia. Induction of proteasome expression by glucocorticoids appears to be a direct result of the downregulation of the activity of nuclear factor ?B, while proteolysis-inducing factor acts through 15-hydroxyeicosatetraenoic acid as an intracellular transducer. Summary: Formation of 15-hydroxyeicosatetraenoic acid is inhibited by eicosapentaenoic acid, which has been shown to attenuate the development of weight loss in patients with pancreatic cancer. When eicosapentaenoic acid is combined with an energy dense nutritional supplement, there is an increase in body weight of cachectic cancer patients through an increase in lean body mass. Eicosapentaenoic acid also prevents protein catabolism and activation of the ubiquitin-proteasome proteolytic pathway during acute starvation in mice, suggesting a similar pathway is involved. Thus eicosapentaenoic acid may be effective in the treatment of protein catabolism in conditions other than cancer.

Raf kinase inhibitor protein RKIP enhances signaling by glycogen synthase kinase-3β

Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor ?B signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3ß (GSK3ß) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3ß protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3ß by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3ß inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and ß-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3ß expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression.