Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.

The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-ß polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS.

Acidic domain in dentin phosphophoryn facilitates cellular uptake: implications in targeted protein delivery.

Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.

Dentin phosphoprotein (DPP) activates integrin-mediated anchorage-dependent signals in undifferentiated mesenchymal cells.

Dentin phosphoprotein (DPP), a major noncollagenous protein of the dentin matrix, is a highly acidic protein that binds Ca(2+) avidly and is thus linked to matrix mineralization. Here, we demonstrate that the RGD domain in DPP can bind to integrins on the cell surface of undifferentiated mesenchymal stem cells and pulp cells. This coupling generates intracellular signals that are channeled along cytoskeletal filaments and activate the non-receptor tyrosine kinase focal adhesion kinase, which plays a key role in signaling at sites of cellular adhesion. The putative focal adhesion kinase autophosphorylation site Tyr(397) is phosphorylated during focal adhesion assembly induced by DPP on the substrate. We further demonstrate that these intracellular signals propagate through the cytoplasm and activate anchorage-dependent ERK signaling. Activated ERK translocates to the nucleus and phosphorylates the transcription factor ELK-1, which in turn coordinates the expression of downstream target genes such as DMP1 and dentin sialoprotein (DSP). These studies suggest a novel paradigm demonstrating that extracellular DPP can induce intracellular signaling that can be propagated to the nucleus and thus alter gene activities.

Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of ß-catenin.

BACKGROUND & AIMS: Pegylated-Interferon-α2a (peg-IFN), a first line therapy for Hepatitis C virus (HCV) patients, also impacts the recurrence of hepatocellular carcinoma (HCC). The activation of the Wnt pathway due to ß-catenin gene mutations contributes to the development of a significant subset of HCC. Herein, we explored the effect of peg-IFN on Wnt/ß-catenin signaling in vitro and in vivo. METHODS: Multiple human hepatoma cell lines were treated with Peg-IFN to assess its effect on the Wnt pathway and the mechanisms involved. Transgenic (TG) mice expressing stable ß-catenin mutant in the liver were exposed to diethylnitrosamine (DEN) and treated with peg-IFN. RESULTS: In vitro, peg-IFN decreased the transcriptional activity of ß-catenin/Tcf and did so independently of JAK/Stat signaling. Peg-IFN treatment led to increased mRNA and protein expression of RanBP3, a known ß-catenin nuclear export factor, in all hepatoma cells. Co-precipitation studies showed an increased association between RanBP3 and ß-catenin after peg-IFN treatment. The siRNA-mediated RanBP3 knockdown abrogated Peg-IFN-induced decrease in TOPFlash reporter activity. In vivo, Peg-IFN treatment led to increased nuclear RanBP3, decreased nuclear ß-catenin and cyclin D1, and decreased cytoplasmic glutamine synthetase. Increased association of RanBP3 and ß-catenin was also observed in vivo in response to Peg-IFN that led to decreased hepatocyte proliferation. CONCLUSIONS: Peg-IFN inhibits ß-catenin signaling through the up-regulation of RanBP3, which may be a contributory mechanism for the delayed HCC and improved survival in treated HCV patients. This observation might have chemo-preventive or chemo-therapeutic implications in tumor with aberrant Wnt pathway activation.

RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage.

Cytopathic viruses have developed successful strategies to block or, at least, to attenuate host interference with their replication. Here, we have analyzed the effects of poliovirus 2A protease on RNA nuclear export. 2A protease interferes with trafficking of mRNAs, rRNAs and U snRNAs from the nucleus to the cytoplasm, without any apparent effect on tRNA transport. Traffic of newly produced mRNAs is more strongly affected than traffic of other mRNAs over-represented in the cytoplasm, such as mRNA encoding beta-actin. Inhibition of RNA nuclear export in HeLa cells expressing 2A protease is concomitant with the cleavage of Nup98, Nup153, Nup62 and their subsequent subcellular redistribution. The expression of an inactive 2A protease failed to interfere with RNA nuclear export. In addition, other related proteases, such as poliovirus 3C or foot and mouth disease virus L(pro) did not affect mRNA distribution or Nup98 integrity. Treatment of HeLa cells with interferon (IFN)-gamma increased the relative amount of Nup98. Under such conditions, the cleavage of Nup98 induced by 2A protease is partial, and thus IFN-gamma prevents the inhibition of RNA nuclear export. Taken together, these results are consistent with a specific proteolysis of Nup98 by 2A protease to prevent de novo mRNA traffic in poliovirus-infected cells.

Terrein reduces pulpal inflammation in human dental pulp cells.

Terrein is a bioactive fungal metabolite whose anti-inflammatory properties are virtually unknown. The purpose of this study was to determine the effects of terrein on lipopolysaccharide (LPS)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human dental pulp cells and to determine the mechanism of the observed effects. The LPS-induced expression of ICAM-1 and VCAM-1 was inhibited by terrein in both a time- and dose-dependent manner. LPS-stimulated translocation of nuclear factor kappa B (NF-kappaB) into the nucleus, which was blocked by inhibitors of amino kinase terminal (AKT, LY294002), extracellular signal regulated kinase 1/2 (ERK 1/2, PD98059), p38 (SB203580), and c-jun NH2-terminal kinase (JNK, SP600125) or terrein. In addition, these inhibitors and terrein also reduced the level of ICAM-1 and VCAM-1 expression in LPS-induced inflammation of pulp cells. Terrein suppressed NF-kappaB activation by blocking the activation of Akt. These results strongly suggest the potential role of terrein as an anti-inflammatory modulator in pulpal inflammation.

Cinnamaldehyde protects human dental pulp cells against oxidative stress through the Nrf2/HO-1-dependent antioxidant response.

Cinnamaldehyde (CA) has various functional properties, such as anti-cancer, anti-microbial, anti-inflammatory, and anti-oxidant activities. This study examined the intracellular signaling mechanisms of CA on the oxidative stress response in human dental pulp cells (hDPCs). The results showed that CA did not have any cell cytotoxicity or cause morphological changes at concentrations up to 50µM. A CA treatment strongly up-regulated the cellular protein level of heme oxygenase-1 (HO-1) and promoted Nrf2 translocation to the nucleus. CA-mediated Nrf2/HO-1 activation reduced the level of reactive oxygen species and protected the hDPCs from H2O2-induced oxidative stress, which induces apoptosis. Moreover, HO-1 depletion by siRNA attenuated the CA-mediated cell protection against oxidative stress. These results indicate that CA protects hDPCs dysfunction under oxidative stress conditions, and this effect is mediated by Nrf2 activation and the up-regulation of HO-1. Overall, these observations suggest that CA is a potential therapeutic agent for cell protection against oxidative stress.

Caffeic acid phenethyl ester attenuates lipopolysaccharide-stimulated proinflammatory responses in human gingival fibroblasts via NF-κB and PI3K/Akt signaling pathway.

Periodontal diseases often begin with chronic gingival inflammation, which causes the destruction of periodontal tissues. Inflammatory immune responses from host cells to bacteria, such as Porphyromonas gingivalis (P. gingivalis), cause periodontal degradation. Human gingival fibroblasts (HGFs) are the major cells in periodontal soft tissues. When stimulated by lipopolysaccharide (LPS), HGFs could secrete several pro-inflammatory cytokines and chemokines, such as interleukins (ILs) IL-6, IL-8, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2). Caffeic acid phenethyl ester (CAPE) is the main active component of propolis, which is collected by honeybees from different plants and known for its anti-inflammatory effects. The anti-inflammatory effects of CAPE on the LPS-induced HGFs were demonstrated in this study. HGFs were pretreated with CAPE (10, 20, and 30µm) for 1h, followed by LPS stimulation (1µg/ml) for 24h. Enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence staining were used to evaluate the production of IL-6, IL-8, iNOS, and COX-2, as well as the activation of TLR4-mediated NF-κB, PI3K/AKT, and MAPK signaling pathways. The results indicated that CAPE inhibits LPS-induced IL-6, IL-8, iNOS, and COX-2 production in a dose-dependent manner. Moreover, CAPE suppresses LPS-induced TLR4/MyD88 and nuclear factor kappa B (NF-κB) activation. In addition, phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) phosphorylation was inhibited by CAPE. These results demonstrated that CAPE could be effective for treating of periodontal diseases.

Effect of BioAggregate on Receptor Activator of Nuclear Factor-Kappa B Ligand-induced Osteoclastogenesis from Murine Macrophage Cell Line In Vitro.

INTRODUCTION: This study aimed to investigate the effect of BioAggregate, a calcium silicate-based nanoparticulate bioceramic, on the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption in vitro, as well as to delineate the underlying molecular mechanism. The performance of BioAggregate was compared with that of ProRoot mineral trioxide aggregate (MTA). METHODS: Cells of a murine macrophage cell line RAW 264.7 were treated with various concentrations of BioAggregate and MTA extracts. Cytotoxicity of material extracts was evaluated with Cell Counting Kit-8 assay. RANKL-induced osteoclast differentiation and function were assessed with tartrate-resistant acid phosphatase staining, F-actin staining, and lacunar resorption pits assay. The mRNA expression associated with osteoclast function was detected with quantitative real-time polymerase chain reaction. Related molecular signaling pathways were investigated with Western blot and immunofluorescence. RESULTS: BioAggregate extracts dose-dependently inhibited RANKL-induced osteoclast formation and resorption capacity without evident cytotoxicity. RAW 264.7 cells exposed to BioAggregate extracts also presented a decrease in RANKL-stimulated mRNA expression of osteoclast-related genes and transcription factors. Moreover, cells treated with BioAggregate extracts exhibited attenuated TRAF6 expression, suppressed mitogen-activated protein kinase signaling, and decreased nuclear translocation of NFATc1 and c-Fos in the presence of RANKL. Comparable effects were induced by MTA. CONCLUSIONS: BioAggregate and MTA exhibit comparable inhibitory effect on osteoclast differentiation and function in vitro, and our findings provide valuable insights into the mechanism of bioceramic-mediated anti-osteoclastogenic activity.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Mechanism of Nanotization-Mediated Improvement in the Efficacy of Caffeine Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism.

The study aimed to measure the neuroprotective efficacy of caffeine-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles over bulk and to delineate the mechanism of improvement in efficacy both in vitro and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinsonism. Caffeine-encapsulated PLGA nanoparticles exhibited more pronounced increase in the endurance of dopaminergic neurons, fibre outgrowth and expression of tyrosine hydroxylase (TH) and growth-associated protein-43 (GAP-43) against 1-methyl-4-phenylpyridinium (MPP+)-induced alterations in vitro. Caffeine-encapsulated PLGA nanoparticles also inhibited MPP(+)-mediated nuclear translocation of nuclear factor-kappa B (NF-κB) and augmented protein kinase B phosphorylation more potentially than bulk counterpart. Conversely, MPTP reduced the striatal dopamine and its metabolites and nigral TH immunoreactivity whereas augmented the nigral microglial activation and nigrostriatal lipid peroxidation and nitrite content, which were shifted towards normalcy by caffeine. The modulations were more evident in caffeine-encapsulated PLGA nanoparticles treated animals as compared with bulk. Moreover, the striatal caffeine and its metabolites were found to be significantly higher in caffeine-encapsulated PLGA nanoparticles-treated mice as compared with bulk. The results thus suggest that nanotization improves the protective efficacy of caffeine against MPTP-induced Parkinsonism owing to enhanced bioavailability, inhibition of the nuclear translocation of NF-κB and activation of protein kinase B phosphorylation.

Nuclear export of L-periaxin, mediated by its nuclear export signal in the PDZ domain.

Myelinating Schwann cells specifically express L-periaxin (L-PRX) in the mammalian peripheral nervous system. Several loss-of-function mutations in periaxin have been described and linked to autosomal recessive Dejerine Sottas neuropathy and to demyelinating Charcot-Marie-Tooth disease. The localization of L-periaxin is developmentally regulated in the nucleus and the plasma membrane of Schwann cells. In this study, L-periaxin, which contains a PDZ domain, a nuclear localization signal (NLS) domain, a repeat domain, and an acidic domain, was localized in the cytoplasm of RSC96 cells. By contrast, a mutant L-periaxin with a deleted PDZ domain was localized mainly in the nucleus of RSC96 cells. After a nuclear cyclin A1, which is localized exclusively in the nucleus, was fused with the PDZ domain, cyclinA1was found in the cytoplasm of RSC96 cells. Treatment with leptomycin B (LMB), a specific inhibitor of nuclear export mediated by leucine-rich nuclear export signal (NES), also causes nuclear accumulation of wild-type L-periaxin. Double leucine mutation (L83, 85Q) in the putative NES in the PDZ domain prevented L-periaxin nuclear export and induced nuclear accumulation. These results suggested that the localization of L-periaxin in the cytoplasm is supported by NES in the PDZ domain.

Non inflammatory boronate based glucose-responsive insulin delivery systems.

Boronic acids, known to bind diols, were screened to identify non-inflammatory cross-linkers for the preparation of glucose sensitive and insulin releasing agglomerates of liposomes (Agglomerated Vesicle Technology-AVT). This was done in order to select a suitable replacement for the previously used cross-linker, ConcanavalinA (ConA), a lectin known to have both toxic and inflammatory effects in vivo. Lead-compounds were selected from screens that involved testing for inflammatory potential, cytotoxicity and glucose-binding. These were then conjugated to insulin-encapsulating nanoparticles and agglomerated via sugar-boronate ester linkages to form AVTs. In vitro, the particles demonstrated triggered release of insulin upon exposure to physiologically relevant concentrations of glucose (10 mmoles/L-40 mmoles/L). The agglomerates were also shown to be responsive to multiple spikes in glucose levels over several hours, releasing insulin at a rate defined by the concentration of the glucose trigger.

Nuclear-cytoplasmic trafficking of NTF2, the nuclear import receptor for the RanGTPase, is subjected to regulation.

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).

New insights on the barrel medic MtOGG1 and MtFPG functions in relation to oxidative stress response in planta and during seed imbibition.

In plants, 8-oxoguanine DNA glycosylase/lyase (OGG1) and formamidopyrimidine-DNA glycosylase (FPG) play similar roles within the base excision repair (BER) pathway involved in the removal of oxidized bases, e.g. 7,8-dihydro-8-oxoguanine (8-oxo-dG) and formamidopyrimidine (FAPy) lesions. To date, it is not clear why plants have retained both the OGG1 and FPG functions. In the present work, we have investigated the possible roles played in planta by MtOGG1 and MtFPG genes from Medicago truncatula Gaertn. (barrel medic). Bioinformatic investigation revealed the presence of putative mitochondrial and nuclear localization signals in the MtOGG1 and MtFPG amino acid sequences, respectively, thus suggesting for different subcellular fates. The expression profiles of both genes were evaluated by Quantitative Real-Time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2), 0.05, 0.1 and 0.2 mM) and polyethylene glycol (PEG6000, 50, 100 and 150 g L(-1)). The MtOGG1 and MtFPG genes were up-regulated in response to stress agents, at different levels, depending on treatment and tissue. As for copper, MtOGG1 showed significant up-regulation (up to 1.2- and 1.7-fold) only in roots while the MtFPG mRNA significantly increased (up to 1.3- and 2.8-fold, respectively) in roots and aerial parts. In response to PEG, the MtOGG1 expression was significantly enhanced in aerial parts (up to 1.3-fold) while the MtFPG showed significant (1.2-fold) up-regulation in roots. The expression profiles of MtOGG1 and MtFPG genes were also evaluated during seed imbibition, a physiological process which is characterized by Reactive Oxygen Species (ROS) accumulation and requires active DNA repair.

Heme oxygenase-1 signals are involved in preferential inhibition of pro-inflammatory cytokine release by surfactin in cells activated with Porphyromonas gingivalis lipopolysaccharide.

Porphyromonas gingivalis is considered the major pathogen of periodontal disease, which leads to chronic inflammation in oral tissues. P. gingivalis-produced lipopolysaccharide (LPS) is a key factor in the development of periodontitis. It is established that surfactin produced by Bacillus subtilis confers anti-inflammatory properties. However, the underlying mechanisms responsible for surfactin-induced anti-inflammatory actions in the context of periodontitis are poorly understood. In this study, we investigated whether surfactin affected P. gingivalis LPS-induced pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-12, and determined that it significantly inhibited their production. Surfactin-mediated inhibition was mainly due to blocked activation of P. gingivalis LPS-triggered nuclear factor-κB. We also examined whether the regulatory effect of surfactin on P. gingivalis LPS-stimulated human THP-1 macrophages was mediated by the induction of heme oxygenase-1 (HO-1) signals, and determined that surfactin also induced HO-1 mRNA and protein expression via activation of Nrf-2. Additionally, we found that small interfering RNA-mediated knock-down of Nrf-2 significantly inhibited surfactin-induced HO-1 expression. Furthermore, inhibition of phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) significantly decreased surfactin-induced HO-1 expression, which is consistent with the suggestion that surfactin-induced HO-1 expression occurs via PI3K/Akt, ERK, and Nrf-2. Treatment with a selective inhibitor of HO-1 reversed the surfactin-mediated inhibition of pro-inflammatory cytokines, suggesting that surfactin induces anti-inflammatory effects by activating Nrf-2-mediated HO-1 induction via PI3K/Akt and ERK signaling. Collectively, these observations support the potential of surfactin as a candidate in strategies to prevent caries, periodontitis, or other inflammatory diseases.

Effect of mineral trioxide aggregate on rat clonal dental pulp cells: expression of cyclooxygenase-2 mRNA and inflammation-related protein via nuclear factor kappa B signaling system.

INTRODUCTION: Recently, mineral trioxide aggregate (MTA) has been routinely used for endodontic treatment. It is well-known that MTA induced secondary dentin formation in pulp cavity when it was applied to dentin, whereas its cytotoxicities were unclear. The purpose of this study was to evaluate the effect of MTA on rat clonal dental pulp cells, RPC-C2A. METHODS: This study was conducted to observe the response of RPC-C2A cells on MTA with reverse-transcriptase polymerase chain reaction, Western blot analysis, and enzyme immunoassay. Data were compared by analysis of variance. Statistical significance was established at P <.01. RESULTS: MTA significantly caused an up-regulation of cyclooxygenase-2 (COX-2) and inducible form of nitric oxide synthase (iNOS) mRNA expression. Furthermore, MTA caused inhibitory kappa B (IkappaB) phosphorylation and translocation of nuclear factor-kappa B (NF-kappaB) subunits to nucleus. Curucumin, an inhibitor of NF-kappaB activation, suppressed MTA-induced COX-2 and iNOS mRNA expressions. In addition, MTA increased the production of prostaglandin E(2) in comparison with the controls. CONCLUSIONS: MTA induces inflammation via NF-kappaB signaling system.

Sevoflurane-mediated TGF-beta1 signaling in renal proximal tubule cells.

Several volatile anesthetics, including sevoflurane, protect against renal ischemia-reperfusion injury in vivo by reducing necrosis and inflammation. Furthermore, in cultured renal tubule cells, sevoflurane directly induced the phosphorylation of the cytoprotective kinases (ERK and Akt), upregulated 70-kDa heat shock protein (HSP70), and attenuated nuclear translocation of the proinflammatory transcription factor NF-kappaB. It has been shown that sevoflurane increases the release of transforming growth factor-beta1 (TGF-beta1) in human proximal tubule (HK-2) cells via externalization of plasma membrane phosphatidylserine (PS), and this increase in TGF-beta1 protected HK-2 cells against hydrogen peroxide-mediated necrosis. In this study, we aimed to determine whether the sevoflurane-mediated phosphorylation of ERK and Akt, induction of HSP70, and reduction in NF-kappaB activation are due to TGF-beta1 receptor-mediated signaling after PS externalization in HK-2 cells. Exogenous TGF-beta1 and a liposome mixture containing PS mimicked sevoflurane-mediated ERK and Akt phosphorylation and HSP70 induction in HK-2 cells. Sevoflurane and TGF-beta1 caused the nuclear translocation of the SMAD3 transcription factor in HK-2 cells. Furthermore, a neutralizing TGF-beta1 antibody or exogenous annexin V to bind PS prevented sevoflurane-induced ERK and Akt phosphorylation and HSP70 induction in HK-2 cells. Finally, a TGF-beta1 antibody and annexin V attenuated the reduction in nuclear translocation of NF-kappaB by sevoflurane. Therefore, we demonstrate in this study that sevoflurane-mediated cytoprotective and anti-inflammatory effects in HK-2 cells are at least partially due to the externalization of PS and activation of TGF-beta1 signaling pathways.

p38 mitogen-activated protein kinase and alkaline phosphatase in human dental pulp cells.

OBJECTIVE: The objective of this study was to investigate the implication of p38 mitogen-activated protein kinase (MAPK) in mediating alkaline phosphatase (ALPase) activity in human dental pulp cells (HPCs). STUDY DESIGN: Nuclear translocation of p38 was observed by immunofluorescence in isolated HPCs treated with transforming growth factor beta1 (TGF-beta1). TGF-beta1 was used to examine the interaction between p38 MAPK and Smad pathway. Role of p38 kinase in mediating ALPase activity was determined with SB203580, a specific inhibitor for the p38 pathway. Lipopolysaccharide (LPS) or TGF-beta1 was added to inhibit or increase ALPase activity. Statistical analysis was performed by unpaired t test. RESULTS: TGF-beta1 induced nuclear translocalization of p38. Blockage of p38 pathway with SB203580 inhibited translocation of Smad2/3 to nuclei. ALPase activity decreased in cells treated with SB203580, in contrast to its vehicle (P < .05). Inhibition on enzyme activity by LPS was exacerbated by SB203580 (P < .05). Treatment with SB203580 before addition of TGF-beta1 also made a significant decrease in ALPase activity (P < .05). CONCLUSIONS: These results suggest that p38 MAPK is implicated in regulating ALPase activity in HPCs and may interact with Smad pathway.

STAT5B-mediated growth hormone signaling is organized by highly dynamic microtubules in hepatic cells.

In the last decade, the notion that microtubules are critical to the spatial organization of signal transduction and contribute to the transmission of signals to downstream targets has been proposed. Because the STAT5B transduction and transcription factor is the major STAT protein activated by growth hormone stimulation in hepatocytes and is a crossroads between many signaling pathways, we studied the involvement of microtubules in STAT5B-mediated growth hormone signaling pathway in the highly differentiated and polarized WIF-B hepatic cell line. We showed that depolymerization of the microtubule network impaired STAT5B translocation to the nucleus upon growth hormone treatment. A significant amount of STAT5B binds to microtubules, while STAT5A and STAT3 are exclusively compartmentalized in the cytosol. Moreover, taxol-induced stabilization of microtubules released STAT5B from its binding, and we show that STAT5B binds specifically to the highly dynamic microtubules and is absent of the stable microtubule subpopulation. The specific involvement of dynamic microtubule subpopulation in growth hormone signaling pathway was confirmed by the inhibition of growth hormone-induced STAT5B nuclear translocation after stabilization of microtubules or specific disruption of highly dynamic microtubules. Upon growth hormone treatment, MT-bound STAT5B was rapidly released from microtubules by a dynein-dependent transport to the nucleus. Altogether, our findings indicate that the labile microtubule subpopulation specifically and dynamically organizes STAT5B-mediated growth hormone signaling in hepatic cells.