Psoriasin has divergent effects on the innate immune responses of murine glial cells.

Antimicrobial peptides are an important part of the innate immune defense in the central nervous system (CNS). The expression of the antimicrobial peptides psoriasin (S100A7) is up-regulated during bacterial meningitis. However, the exact mechanisms induced by psoriasin to modulate glial cell activity are not yet fully understood. Our hypothesis is that psoriasin induced pro- and anti-inflammatory signaling pathways as well as regenerative factors to contribute in total to a balanced immune response. Therefore, we used psoriasin-stimulated glial cells and analyzed the translocation of the pro-inflammatory transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) in murine glial cells and the expression of pro- and anti-inflammatory mediators by real time RT-PCR, ELISA technique, and western blotting. Furthermore, the relationship between psoriasin and the antioxidative stress transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) was investigated. Stimulation with psoriasin not only enhanced NFκB translocation and increased the expression of the pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) but also neurotrophin expression. Evidence for functional interactions between psoriasin and Nrf2 were detected in the form of increased antioxidant response element (ARE) activity and induction of Nrf2/ARE-dependent heme oxygenase 1 (HO-1) expression in psoriasin-treated microglia and astrocytes. The results illustrate the ability of psoriasin to induce immunological functions in glia cells where psoriasin exerts divergent effects on the innate immune response.

In vivo imaging of antioxidant response element activity during liver regeneration after partial hepatectomy.

BACKGROUND: The nuclear factor-erythroid 2-related factor 2 (Nrf2) -antioxidant response element (ARE) pathway is important for the regulation of antioxidative stress response and detoxification. To activate the expression of its target genes, such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1), Nrf2 binds to the ARE within the promoter region of these genes. Partial hepatectomy and consecutive liver regeneration lead to oxidative stress with activation of the Nrf2-ARE pathway. The aim of this study was to investigate ARE activity in vivo during liver regeneration after partial hepatectomy. MATERIALS AND METHODS: Transgenic ARE-luc mice were used. In these mice, the luciferase reporter gene is under the control of an ARE promoter element. Following 2/3 partial hepatectomy (PHx), mice underwent in vivo bioluminescence imaging up until the ninth postoperative day. In addition, liver tissue was analyzed by immunohistochemistry (Nrf2 and HO-1), quantitative reverse transcription-PCR (HO-1 and NQO1) and in vitro luminescence assays. RESULTS: Bioluminescence imaging revealed a significant increase in Nrf2-ARE activity after PHx. The signal maximum was recorded on the third day after PHx. Seven days postoperatively, the signal almost reached baseline levels. In immunohistochemistry, significantly more hepatocytes were positive for Nrf2 and HO-1 on the third postoperative day compared with baseline levels. The mRNA expression of HO-1 and NQO1 were significantly increased on day 3 as measured by qRT-PCR. CONCLUSIONS: This study demonstrated the time-dependent activation of the Nrf2-ARE system during liver regeneration in vivo. The transgenic ARE-luc mouse provided a convenient model for studying Nrf2-mediated gene expression noninvasively and may facilitate further experiments with therapeutic modulation of the antioxidative stress response.

Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development.

Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible "second hit" triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1(Δhepa)) and littermate Cre- controls (Keap1(fx/fx)) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1(Δhepa) mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1(Δhepa) compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/ß) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1(Δhepa) livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1(Δhepa) compared to Keap1(fx/fx) as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.

Mechanical forces induce changes in VEGF and VEGFR-1/sFlt-1 expression in human chondrocytes.

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.

LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.

Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE)--and amyloid beta 1-42-induced signal transduction in glial cells.

BACKGROUND: Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer's disease (AD).Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-ß 1-42 (Aß1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains. RESULTS: We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aß1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy. CONCLUSIONS: The results suggest that both formyl peptide receptors play an essential role in Aß1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.

Impact of Nrf2 on esophagus epithelium cornification.

BACKGROUND: Nrf2 is a transcription factor that is known to maintain cellular defense against the toxicity of electrophiles and reactive oxygen species (ROS). METHODS: We show an effect of Nrf2 deficiency on the histology of the esophagus of Nrf2 knockout mice. RESULTS: Quantitative analysis of esophageal cornification via hematoxylin and eosin (H&E) staining revealed significantly (P=0.0127) decreased stratification in Nrf2 knockout mice compared with wild-type controls. In addition, we show that Nrf2 is expressed solely in the stratum spinosum and not in the stratum basale of the epidermis. This expression pattern is exactly the same as those described for keratin K6 and K16. CONCLUSIONS: We conclude that Nrf2 regulates the cornification of epithelia and may play a role in callus formation and wound healing of the skin, as well as in skin aging.

Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals.

Allergic contact dermatitis is induced by repeated skin contact with an allergen. Assessment of the skin sensitizing potential of chemicals, agrochemicals, and especially cosmetic ingredients is currently performed with the use of animals. Animal welfare and EU legislation demand animal-free alternatives reflected in a testing and marketing ban for cosmetic ingredients beginning in 2013. The underlying mechanisms of induction and elicitation of skin sensitization are complex and a chemical needs to comply several properties being skin sensitizing. To account for the multitude of events in the induction of skin sensitization an in vitro test system will consist of a battery of various tests. Currently, we performed intralaboratory validations of four assays addressing three different events during induction of skin sensitization. (1) The Direct Peptide Reactivity Assay (DPRA) according to Gerberick and co-workers (Gerberick et al., 2004) using synthetic peptides and HPLC analysis. (2) Two dendritic cell activation assays based on the dendritic cell like cell lines U-937 and THP-1 and flow cytometric detection of the maturation markers CD54 and/or CD86 (Ashikaga et al., 2006; Python et al., 2007; Sakaguchi et al., 2006). (3) Antioxidant response element (ARE)-dependent gene activity in a HaCaT reporter gene cell line (Emter et al., 2010). We present the results of our intralaboratory validation of these assays with 23 substances of known sensitizing potential. The sensitivity, specificity, and accuracy of the individual tests were obtained by comparison to human epidemiological data as well as to data from animal tests such as the local lymph node assay.

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis.

BACKGROUND: Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. METHODS: Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined. RESULTS: We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1ß expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction. CONCLUSIONS: We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.

Antimicrobial peptide rCRAMP induced glial cell activation through P2Y receptor signalling pathways.

Antimicrobial peptides are part of the innate immune system of many organ systems, yet little is known about their expression and function in the brain. The antibacterial cathelicidin rCRAMP in rats (homologue of the human LL-37) not only exhibits potent bactericidal activities but also functions as a chemoattractant for immune cells. In this study, to further evaluate the role of rCRAMP in innate immunity of brain cells, we investigated the impact of rCRAMP on glial cell activation. To this end we analyzed the activation of rCRAMP-induced signalling by cytokine expression, Western blotting of certain signal transduction pathways and by cAMP level measurement in primary rat glial cells (astrocytes and microglia). We demonstrate (i) the induction of proinflammatory cytokine and neurotrophic factors and (ii) the activation of various signal transduction pathways by rCRAMP in glial cells. Moreover, (iii) we have been able to show that rCRAMP-induced IL-6 expression and ERK1/2 phosphorylation in glial cells were attenuated by the antagonists for purinergic receptor P2Y, whereas P2X and FPRL1 antagonists do not show any effects. Our results indicate for the first time that a newly identified P2Y11 receptor participates in rCRAMP-induced signal transduction. This study provides evidence that rCRAMP participates in brain immunity by stimulating cytokine production and glial cell activation, and aid in the protection of brain cells by inducing neurotrophic factors.

ADAM12 is expressed by astrocytes during experimental demyelination.

A disintegrin and metalloproteinase (ADAM) 12 represents a member of a large family of similarly structured multi-domain proteins. In the central nervous system (CNS), ADAM12 has been suggested to play a role in brain development, glioblastoma cell proliferation, and in experimental autoimmune encephalomyelitis. Furthermore, ADAM12 was reported to be almost exclusively expressed by oligodendrocytes and could, therefore, be considered as suitable marker for this cell type. In the present study, we investigated ADAM12 expression in the healthy and pathologically altered murine CNS. As pathological paradigm, we used the cuprizone demyelination model in which myelin loss during multiple sclerosis is imitated. Besides APC(+) oligodendrocytes, SMI311(+) neurons and GFAP(+) astrocytes express ADAM12 in the adult mouse brain. ADAM12 expression was further analyzed in vitro. After the induction of demyelination, we observed that activated astrocytes are the main source of ADAM12 in brain regions affected by oligodendrocyte loss. Exposure of astrocytes in vitro to either lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFalpha), glutamate, or hydrogen peroxide revealed a highly stimulus-specific regulation of ADAM12 expression which was not seen in microglial BV2 cells. It appears that LPS- and TNFalpha-induced ADAM12 expression is mediated via the classic NFkappaB pathway. In summary, we demonstrated that ADAM12 is not a suitable marker for oligodendrocytes. Our results further suggest that ADAM12 might be implicated in the course of distinct CNS diseases such as demyelinating disorders.

Functional and physical interactions between formyl-peptide-receptors and scavenger receptor MARCO and their involvement in amyloid beta 1-42-induced signal transduction in glial cells.

Recent studies suggest that the chemotactic G protein-coupled receptor formyl-peptide-receptor-like-1 (FPRL1) or the scavenger receptor MARCO (macrophage receptor with collagenous structure) plays an essential role in the inflammatory response of host defense mechanisms and neurodegenerative disorders such as Alzheimer's disease. We therefore analyzed the involvement of FPRL1 and MARCO in amyloid beta1-42 (Abeta1-42)-induced signalling by extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and in transfected HEK293 cells. Receptors were inhibited by small interference RNA and the consequences in Abeta1-42- and MARCO agonist fucoidan-induced signal transduction were determined. Receptor deactivation by antagonists or small interference RNA verified the importance of FPRL1 for Abeta1-42-mediated signal transduction by ERK1/2 phosphorylation and cAMP level measurement in glial cells. Furthermore, for the first time, we have demonstrated a functional interaction between FPRL1 and scavenger receptors in fucoidan-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition, co-immunoprecipitation data and fluorescence microscopy measurements revealed a physical interaction between FPR, FPRL1 and MARCO. These results suggest that FPRL1 plays a pivotal role for Abeta1-42-induced signal transduction in glial cells and the interaction with MARCO could explain the broad ligand spectrum of formyl peptide receptors.

Sulforaphane suppresses LPS-induced inflammation in primary rat microglia.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the signal transduction pathways involved in sulforaphane (SF) mediated inhibition of the inflammatory response to lipopolysaccharide (LPS). Additionally, we investigated the effects of SF and LPS on the activity of Nrf2. MATERIAL: Primary rat microglia and the murine microglia cell line BV2 were used. TREATMENT: Cells were treated with LPS with or without SF. METHODS: Cell viability was measured via WST-assay. Real-time RT-PCR was performed to analyze cytokine mRNA levels. The nitric oxide (NO) release was measured in LPS-stimulated microglia. The induction of various signal transduction pathways and Nrf2 was determined by Western blotting. NF-kappaB and AP-1 activation was measured by dual luciferase assay. RESULTS: We showed that SF attenuates the LPS-induced increase of IL-1beta, IL-6, and TNF-alpha expression in microglia. In addition, SF significantly decreases the NO in a concentration-dependent manner. SF inhibits LPS-stimulated ERK1/2 and JNK phosphorylation and thereby inhibits the LPS-induced activation of NF-kappaB- and activator protein-1 (AP-1). Moreover, SF and LPS together are able to induce Nrf2 activation. CONCLUSIONS: We showed that SF, and also LPS by itself, are able to activate the cell's defence against oxidative and electrophilic stress. We conclude that SF could be a candidate agent for anti-inflammatory treatment of the central nervous system.

Expression and regulation of antimicrobial peptide rCRAMP after bacterial infection in primary rat meningeal cells.

Bacterial meningitis is characterized by an inflammation of the meninges and continues to be an important cause of mortality and morbidity. Meningeal cells cover the cerebral surface and are involved in the first interaction between pathogens and the brain. Little is known about the role of meningeal cells and the expression of antimicrobial peptides in the innate immune system. In this study we characterized the expression, secretion and bactericidal properties of rat cathelin-related antimicrobial peptide (rCRAMP), a homologue of the human LL-37, in rat meningeal cells after incubation with different bacterial supernatants and the bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). Using an agar diffusion test, we observed that supernatants from meningeal cells incubated with bacterial supernatants, LPS and PGN showed signs of antimicrobial activity. The inhibition of rCRAMP expression using siRNA reduced the antimicrobial activity of the cell culture supernatants. The expression of rCRAMP in rat meningeal cells involved various signal transduction pathways and was induced by the inflammatory cytokines interleukin-1, -6 and tumor necrosis factor alpha. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae and rCRAMP was localized in meningeal cells using immunohistochemistry. These results suggest that cathelicidins produced by meningeal cells play an important part in the innate immune response against pathogens in CNS bacterial infections.

Internalization and signal transduction of PrP(106-126) in neuronal cells.

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood steps in the pathogenesis of prion diseases. We have thus analyzed the internalization and signal transduction of the neurotoxic fragment of the prion protein PrP(106-126) in the rat neuroblastoma cell line B104 by fluorescence microscopy and quantification by ELISA and in primary neuronal cells from mice. Phospholipase D (PLD) is known to be an enzyme involved in the regulation of secretion, endocytosis and receptor signalling. We determined the PLD activity using a transphosphatidylation assay and could show that PLD is involved in PrP(106-126) internalization. The determination of receptor activity via quantification of ERK1/2 phosphorylation and cAMP level measurement verified the PrP(106-126)-induced signal transduction in B104 cells and primary neuronal cells. PrP(106-126)-induced a decrease in cAMP level in neuronal cells. These studies indicate the involvement of PLD in PrP(106-126)-endocytosis and mediated cellular signalling by an unidentified inhibitory G-protein-coupled receptor and may allow the development of therapeutic agents interfering with prion uptake and/or PLD function using PLD as a possible pharmaceutical target.

Role of glial cells in the functional expression of LL-37/rat cathelin-related antimicrobial peptide in meningitis.

Antimicrobial peptides are intrinsic to the innate immune system in many organ systems, but little is known about their expression in the central nervous system. We examined cerebrospinal fluid (CSF) and serum from patients with active bacterial meningitis to assess antimicrobial peptides and possible bactericidal properties of the CSF. We found antimicrobial peptides (human cathelicidin LL-37) in the CSF of patients with bacterial meningitis but not in control CSF. We next characterized the expression, secretion, and bactericidal properties of rat cathelin-related antimicrobial peptide, the homologue of the human LL-37, in rat astrocytes and microglia after incubation with different bacterial components. Using real-time polymerase chain reaction and Western blotting, we determined that supernatants from both astrocytes and microglia incubated with bacterial component supernatants had antimicrobial activity. The expression of rat cathelin-related antimicrobial peptide in rat glial cells involved different signal transduction pathways and was induced by the inflammatory cytokines interleukin 1beta and tumor necrosis factor. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae, and rat cathelin-related antimicrobial peptide was localized in glia, choroid plexus, and ependymal cells by immunohistochemistry. Together, these results suggest that cathelicidins produced by glia and other cells play an important part in the innate immune response against pathogens in central nervous system bacterial infections.

Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation.

One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide (AP), which can initiate a cascade of oxidative events that may result in neuronal death. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is the major regulator for a battery of genes encoding detoxifying and antioxidative enzymes via binding to the antioxidant response element (ARE), it is of great interest to find nontoxic activators of Nrf2 rendering neuronal cells more resistant to AP toxicity. Using ARE-luciferase assay and Western blot, we provide evidence that the kavalactones methysticin, kavain, and yangonin activate Nrf2 time- and dose-dependently in neural PC-12 and astroglial C6 cells and thereby up-regulate cytoprotective genes. Viability and cytotoxicity assays demonstrate that Nrf2 activation is able to protect neural cells from amyloid beta-(1-42) induced neurotoxicity. Down-regulation of Nrf2 by small hairpin RNA as well as extracellular signal-regulated kinase 1/2 inhibition abolishes cytoprotection. We further give evidence that kavalactone-mediated Nrf2 activation is not dependent on oxidative stress production. Our results demonstrate that kavalactones attenuate amyloid beta-peptide toxicity by inducing protective gene expression mediated by Nrf2 activation in vitro. These findings indicate that the use of purified kavalactones might be considered as an adjunct therapeutic strategy to combat neural demise in Alzheimer disease and other oxidative stress-related diseases.

Programmable cells of monocytic origin (PCMO): a source of peripheral blood stem cells that generate collagen type II-producing chondrocytes.

The focus of this study was a new adult pluripotent cell derived from human peripheral blood monocytes identified as a "programmable cell of monocytic origin" (PCMO). In contrast to bone marrow-derived stem cells, these cells can be harvested from peripheral venous blood without aspiration of the bone marrow and have multilineage potential comparable to that of mesenchymal stem cells (MSC). The aim of this study was to evaluate the potential of PCMOs to differentiate into collagen type II-producing chondrocytes using various extrinsic cues (TGFbeta-1, IGF-1, BMP-2, and BMP-7). Collagen type I and II proteins were localized using immunohistochemistry and quantified by enzyme-linked immunosorbent assays (ELISA). The shape of the differentiating PCMOs was monitored with electron microscopy. Collagen type I and II messenger RNA expression was analyzed using real-time reverse transcriptase-polymerase chain reaction (RT PCR) and regular RT PCR. Immunohistochemistry revealed a strong accumulation of collagen type II after a 6-week incubation period with BMP-2, BMP-7, TGF-beta, IGF-I, and TGF-beta, and IGF-1. Collagen type I was only mildly induced by the applied stimulants. Electron microscopy findings showed a shift from a monocyte-like structure to a chondrocyte-like structure after 2 weeks of stimulation. Stimulation of PCMOs with BMP-2, BMP-7, TGF-beta, IGF-I, and TGF-beta, and IGF-1 induced a chondrogenic differentiation with continuous expression of collagen type II mRNA and protein over several weeks time. Collagen type I and II expression in undifferentiated PCMOs or in control cells incubated without any stimulant was not detected. PCMOs have the potential to differentiate into collagen type II synthesizing chondrocytes. The ability to reprogram and differentiate PCMOs from peripheral blood into sizable quantities might enable their clinical application in cartilage repair after mechanical injury or in cases of osteoarthritis.

Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants.

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0-20 microg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0-20 microM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR. Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium. Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.

Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein.

OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface. METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal. Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.