Concomitant Phosphorylation Research Articles

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK

Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58IPK. In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, β1-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (P<0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D2 is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58IPK expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58IPK by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58IPK expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58IPK prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58IPK upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.

Chk2 mediates RITA-induced apoptosis

Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.

Ecdysone receptor and ultraspiracle proteins are tyrosine phosphorylated during adult development of silkmoths

20-hydroxy ecdysone (20E) is essential to promote adult development in diapausing silkmoth pupae. Increases in protein tyrosine/serine-phosphorylations observed soon after 20E administration supported the initial hypothesis that activation of receptor tyrosine kinase-ras-MAPK pathway could be responsible for the growth promoting effects of 20E. This report pertains to the high levels of protein tyrosine phosphorylations (PTP) that occurred later during the growth to differentiation transition because of its novelty and relevance to 20E dependence of adult development. Further analyses demonstrated that both ecdysone receptor (EcR) and ultraspiracle (USP), the two dimerizing partners of the functional ecdysone receptor, are tyrosine phosphorylated coincidental with high PTP. Enhanced PTP during growth to differentiation transition and concomitant tyrosine phosphorylation of EcR and USP was shown to occur in another silkmoth species pointing to the necessity of similar protein tyrosine phosphorylation pathways for adult development. Properly timed increases in tissue protein tyrosine kinase (PTK) activity could explain the enhancement of PTP in the wing epidermis of both the silkmoths. Thymidine incorporation measurements showed that cessation of DNA synthesis preceded the increase in PTK activity thus emphasizing a role for PTP in aspects of tissue physiology related to differentiative events rather than cell proliferation. Phosphatase and tyrosine kinase inhibitors (Tyrphostins) had minimal effects on adult wing development in vivo. However, the escape of the adult from the pupal case was blocked by a tyrphostin indicating the importance of PTKs in eclosion.

Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy

The mannitol transporter EIImtl from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EIImtl is functional as a dimer and its membrane-embedded C domain, IICmtl, harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IICmtl. At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IICmtl. Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EIImtl is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IICmtl structure are involved in the sugar translocation. The structure of EIImtl, as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EIImtl, the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.

Developmental localization of NMDA receptors, Src and MAP kinases in mouse brain

Activation of NMDA receptors (NMDAR) is associated with divergent downstream signaling leading to neuronal survival or death that may be regulated in part by whether the receptor is located synaptically or extrasynaptically. Distinct activation of the MAP kinases ERK and p38 by synaptic and extrasynaptic NMDAR is one of the mechanisms underlying these differences. We have recently shown that the Src family kinases (SFKs) play an important role in neonatal hypoxic–ischemic brain injury by regulating NMDAR phosphorylation. In this study, we characterized the distribution of NMDAR, SFKs and MAP kinases in synaptic and extrasynaptic membrane locations in the postnatal day 7 and adult mouse cortex. We found that the NMDAR, SFKs and phospho-NR2B were predominantly at synapses, whereas striatal-enriched protein tyrosine phosphatase (STEP) and its substrates ERK and p38 were much more concentrated extrasynaptically. NR1/NR2B was the main subunit at extrasynaptic membrane with concomitant NR2B phosphorylation at tyrosine (Y) 1336 in the immature brain. STEP expression increased, while p38 decreased with development in the extrasynaptic membrane. These results suggest that SFKs and STEP are poised to differentially regulate NMDAR-mediated signaling pathways due to their distinct subcellular localization, and thus may contribute to the age-specific differences seen in vulnerability, pathology and consequences of hypoxic–ischemic brain injury.

Ligation of CD24 expressed by oral epithelial cells induces kinase dependent decrease in paracellular permeability mediated by tight junction proteins

In previous studies we demonstrated uniform strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies auto-reactive with CD24 peptide correlated with reduced severity of periodontal disease. In the present study an epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Ligation of CD24 expressed by oral epithelial cells with an anti-CD24 antibody induced formation of tight junctions and live-cell imaging confirmed that paracellular diffusion of fluorochrome-labeled dextran was reduced. Expression of mRNA and protein for zona occludens-1, -2, junction adhesion molecule-A (JAM-A), occludin and claudins-1, -4, -8, -15, -18 was significantly increased following ligation of CD24 but only claudins-4 and -15, JAM-A, occludin and zona occludens-1 and -2 were increased at cell contacts. This change in expression patterns reflected that observed between the epithelium of the periodontal lesion and that of the healthy gingival attachment. In the model system, response profiles to kinase inhibitors indicated a key role of c-Src kinase activation in the development of diffusion-limiting tight junction complexes. Activation was confirmed by demonstrating concomitant phosphorylation of the kinase. Pre-incubation with antibodies against JAM-A and claudin-15 prevented barrier-enhancing effects of anti-CD24 antibodies while pre-incubation with antibody to claudin-4 was partially effective. It is concluded that antibodies to CD24 facilitate expression and location of JAM-A, claudins-4 and -15 that mediate enhanced epithelial barrier function in a protective response against bacterial products.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-d-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1h. While 24h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Mitochondrial Localized STAT3 Is Involved in NGF Induced Neurite Outgrowth

BackgroundSignal transducer and activator of transcription 3 (STAT3) plays critical roles in neural development and is increasingly recognized as a major mediator of injury response in the nervous system. Cytokines and growth factors are known to phosphorylate STAT3 at tyrosine705 with or without the concomitant phosphorylation at serine727, resulting in the nuclear localization of STAT3 and subsequent transcriptional activation of genes. Recent evidence suggests that STAT3 may control cell function via alternative mechanisms independent of its transcriptional activity. Currently, the involvement of STAT3 mono-phosphorylated at residue serine727 (P-Ser-STAT3) in neurite outgrowth and the underlying mechanism is largely unknown.Principal FindingsIn this study, we investigated the role of nerve growth factor (NGF) induced P-Ser-STAT3 in mediating neurite outgrowth. NGF induced the phosphorylation of residue serine727 but not tyrosine705 of STAT3 in PC12 and primary cortical neuronal cells. In PC12 cells, serine but not tyrosine dominant negative mutant of STAT3 was found to impair NGF induced neurite outgrowth. Unexpectedly, NGF induced P-Ser-STAT3 was localized to the mitochondria but not in the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NGF induced neurite outgrowth and the production of reactive oxygen species (ROS).ConclusionTaken together, the findings herein demonstrated a hitherto unrecognized novel transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 is involved in NGF induced neurite outgrowth.

Olanzapine-Induced Hyperphagia and Weight Gain Associate with Orexigenic Hypothalamic Neuropeptide Signaling without Concomitant AMPK Phosphorylation

The success of antipsychotic drug treatment in patients with schizophrenia is limited by the propensity of these drugs to induce hyperphagia, weight gain and other metabolic disturbances, particularly evident for olanzapine and clozapine. However, the molecular mechanisms involved in antipsychotic-induced hyperphagia remain unclear. Here, we investigate the effect of olanzapine administration on the regulation of hypothalamic mechanisms controlling food intake, namely neuropeptide expression and AMP-activated protein kinase (AMPK) phosphorylation in rats. Our results show that subchronic exposure to olanzapine upregulates neuropeptide Y (NPY) and agouti related protein (AgRP) and downregulates proopiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC). This effect was evident both in rats fed ad libitum and in pair-fed rats. Of note, despite weight gain and increased expression of orexigenic neuropeptides, subchronic administration of olanzapine decreased AMPK phosphorylation levels. This reduction in AMPK was not observed after acute administration of either olanzapine or clozapine. Overall, our data suggest that olanzapine-induced hyperphagia is mediated through appropriate changes in hypothalamic neuropeptides, and that this effect does not require concomitant AMPK activation. Our data shed new light on the hypothalamic mechanism underlying antipsychotic-induced hyperphagia and weight gain, and provide the basis for alternative targets to control energy balance.

The phospho enolpyruvate-dependent glucose–phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell

The phospho enolpyruvate-(PEP)-dependent-carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex transport and sensory system. Such a PTS usually consists of two cytoplasmic energy-coupling proteins, Enzyme I (EI) and HPr, and one of more than 20 different carbohydrate-specific membrane proteins named Enzyme II (EII), which catalyze the uptake and concomitant phosphorylation of numerous carbohydrates. The most prominent representative is the glucose–PTS, which uses a PTS-typical phosphorylation cascade to transport and phosphorylate glucose. All components of the glucose–PTS interact with a large number of non-PTS proteins to regulate the carbohydrate flux in the bacterial cell. Several aspects of the glucose–PTS have been intensively investigated in various research projects of many groups. In this article we will review our recent findings on a Glc-PTS-dependent metalloprotease, on the interaction of EIICB Glc with the regulatory peptide SgrT, on the structure of the membrane spanning C-domain of the glucose transporter and on the modeling approaches of ptsG regulation, respectively, and discuss them in context of general PTS research.

Inhibition of glioblastoma growth by the thiadiazolidinone compound TDZD-8.

BackgroundThiadiazolidinones (TDZD) are small heterocyclic compounds first described as non-ATP competitive inhibitors of glycogen synthase kinase 3β (GSK-3β). In this study, we analyzed the effects of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), on murine GL261 cells growth in vitro and on the growth of established intracerebral murine gliomas in vivo.Methodology/Principal FindingsOur data show that TDZD-8 decreased proliferation and induced apoptosis of GL261 glioblastoma cells in vitro, delayed tumor growth in vivo, and augmented animal survival. These effects were associated with an early activation of extracellular signal-regulated kinase (ERK) pathway and increased expression of EGR-1 and p21 genes. Also, we observed a sustained activation of the ERK pathway, a concomitant phosphorylation and activation of ribosomal S6 kinase (p90RSK) and an inactivation of GSK-3β by phosphorylation at Ser 9. Finally, treatment of glioblastoma stem cells with TDZD-8 resulted in an inhibition of proliferation and self-renewal of these cells.Conclusions/SignificanceOur results suggest that TDZD-8 uses a novel mechanism to target glioblastoma cells, and that malignant progenitor population could be a target of this compound.

Spatial and temporal organization of the E. coli PTS components

The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to β-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E 2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E 2. The present results suggest that BPA, like 17β-E 2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E 2 when it coexists with 17β-E 2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

2-Arylthiazolidine-4-carboxylic acid amides (ATCAA) target dual pathways in cancer cells: 5'-AMP-activated protein kinase (AMPK)/mTOR and PI3K/Akt/mTOR pathways

Phosphatidylinositol-3-kinase (PI3K)/Akt and 5'-AMP-activated protein kinase (AMPK) are attractive targets for anti-cancer drug development. Inhibition of Akt or activation of AMPK is cytotoxic to human cancer cells in vitro and in vivo. We previously demonstrated that 2-arylthiazolidine-4-carboxylic acid amides (ATCAA) are effective cytotoxic agents in prostate and melanoma cancer cell lines, with IC(50) values in the low/sub micromolar range. Using in vitro and in vivo studies, we further characterized the anti-cancer efficacy and mechanism of action of ATCAA-10, a potent lead. ATCAA-10 exhibited equal potency on both MES/SA and P-glycoprotein over-expressing multidrug resistant MES/SA/Dx5 cells, suggesting that ATCAA-10 may overcome multiple drug resistance. Cell-free kinase binding assays excluded the direct binding of ATCAA-10 to several kinases, including IGF-1R, EGFR, FGFR and PDGFR. However, in A549 and HeLa cells, ATCAA-10 effectively dephosphorylated Akt, with concomitant phosphorylation of AMPK. Determination of intracellular ATP and AMP concentrations revealed that ATCAA-10 activated AMPK by altering the intracellular AMP/ATP ratio. ATCAA-10 exhibited favorable pharmacokinetic properties in both mice and rats, including low clearance, low hepatic extraction rate, moderate volume of distribution and long half-life. In addition, ATCAA-10 inhibited A549 tumor xenograft growth with 46% tumor growth inhibition (TGI) at 20 mg/kg dose. Taken together; these results suggest that ATCAA-10 modulates the activity of two signaling pathways, PI3K/AKT/mTOR and AMPK/mTOR, resulting in the inhibition of cancer cell growth.

Abstract 3181: Connecting EGFR activation to inflammatory signaling in epithelial ovarian cancer

Abstract Epithelial Ovarian Cancer (EOC) is the leading cause of death from gynecology cancer. EOC is initially characterized by intraperitoneal growth of tumor masses followed by an increased accumulation of ascitic fluid that contains a variable numbers of tumor cells and inflammatory leukocytes, as well as, variable levels of cytokines and chemokines. The receptor/ligand pair CXCR4/CXCL12 and the cytokines IL-1β, IL-6 and IL-8 have been found to contribute to cell proliferation of EOC. Despite our limited knowledge of initiating events of EOC, it is known that RAS oncogene mutation account for 30% of advanced EOC, activated H-RAS protein is commonly detected in EOC and activation of a receptor tyrosine kinase (RTK) could be the upstream mechanism initiating the cascade via the RAS/mitogen-activated protein kinases that lead to transcriptional activation. Indeed, in a homeostatic state EGFR, member of HER family RTK, plays a key role in normal ovarian follicle development and in the growth regulation of cells of ovarian surface epithelium (OSE) from which the EOC arises. EGFR is expressed in an estimated 35% to 70% of EOCs. Here, we wanted to assess in EOC whether the activation of the oncogene EGFR is related to an inflammation-associated signaling. Within normal and several transformed ovarian cell lines we selected IGROV-1 and OAW42 cells showing the highest EGFR expression and IL-6 release. Upon receptor activation with EGF, an up-regulation of PI3K/AKT and MEK/ERK pathways were observed. In both cell lines phosphorylation of EGFR occurred in the first 15 min of EGF treatment, followed by a concomitant phosphorylation of both AKT and MAPK. We also observed that the cited phosphorylations were completely inhibited by using the AG1478 EGFR inhibitor. In both cell lines EGF triggered NF-kB transcriptional activation as evaluated by transient transfection with a promoter-reporter gene construct. In the EOC selected cells, upon EGFR activation, an up-regulation of IL-6 mRNA was observed and immunofluorescence analysis revealed that NF-kB translocated into the nucleus. To identify the secretion of specific inflammatory molecules induced by EGFR/NFkB activation in EOC cells, a fifty-one cyto-/chemokine panel in conditioned media from EGF-treated cells have been analysed by Bioplex system. We have identified some inflammatory molecules released specifically upon EGFR activation in a time dependent way such as IL-6, PAI-1, IL-5, IL-12 and VEGF. On a limited number of ascitic fluids from EOC patients IL-6 and PAI-1 have been also analysed by ELISA assay, and the correlation with the EGFR expression on tumor cells of the same patients will be carried out. Overall the acquired information about the inflammation-related EGFR could lead to the identification of alternative approaches potentially suited for improving diagnosis and treatment of EOCs. Partially supported by Italian Health Ministry (Grant ACC1) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3181.

Aberrant activation of STAT3 in primary human endothelial cells by the latent KSHV protein kaposin B (39.18)

Abstract The proto-oncogene STAT3 is a latent transcription factor that upon activation drives the expression of a number of genes involved in cell proliferation, survival, and immune responses. Canonical STAT3 activation occurs via phosphorylation of Y705, dimerization, and nuclear translocation, followed by phosphorylation of S727 for maximal transcriptional activity. Kaposi’s sarcoma associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers including Kaposi’s sarcoma (KS). Here we show that latent KSHV infection of primary human endothelial cells (ECs) activates STAT3, and identify a key latency protein, kaposin B, that contributes to this activation. Kaposin B expression in ECs causes STAT3 phosphorylation at S727, relocalization to punctate structures within the nucleus known as ‘STAT3 bodies’, and enhanced expression of STAT3 target genes. Recent work shows that the tripartite motif-containing protein 28 (TRIM28, a.k.a. TIF-1β, KAP-1) negatively regulates STAT3 by recruiting transcriptional silencing complexes. Here, we demonstrate for the first time that TRIM28 is a bona fide target of the host cell kinase MK2 and show that KSHV infection and kaposin B expression results in MK2 activation and concomitant phosphorylation and inactivation of TRIM28. Taken together, our studies reveal an important new regulatory mechanism governing STAT3 activation in endothelial cells and suggest novel protein targets for the treatment of KS.

The Fibroblast Integrin α11β1 Is Induced in a Mechanosensitive Manner Involving Activin A and Regulates Myofibroblast Differentiation

Fibrotic tissue is characterized by an overabundance of myofibroblasts. Thus, understanding the factors that induce myofibroblast differentiation is paramount to preventing fibrotic healing. Previous studies have shown that mechanical stress derived from the integrin-mediated interaction between extracellular matrix and the cytoskeleton promotes myofibroblast differentiation. Integrin alpha11beta1 is a collagen receptor on fibroblasts. To determine whether alpha11beta1 can act as a mechanosensor to promote the myofibroblast phenotype, mouse embryonic fibroblasts and human corneal fibroblasts were utilized. We found that alpha11 mRNA and protein levels were up-regulated in mouse embryonic fibroblasts grown in attached three-dimensional collagen gels and conversely down-regulated in cells grown in floating gels. alpha11 up-regulation could be prevented by manually detaching the collagen gels or by cytochalasin D treatment. Furthermore, SB-431542, an inhibitor of signaling via ALK4, ALK5, and ALK7, prevented the up-regulation of alpha11 and the concomitant phosphorylation of Smad3 under attached conditions. In attached gels, TGF-beta1 was secreted in its inactive form but surprisingly not further activated, thus not influencing alpha11 regulation. However, inhibition of activin A attenuated the up-regulation of alpha11. To determine the role of alpha11 in myofibroblast differentiation, human corneal fibroblasts were transfected with small interfering RNA to alpha11, which decreased alpha-smooth muscle actin expression and myofibroblast differentiation. Our data suggest that alpha11beta1 is regulated by cell/matrix stress involving activin A and Smad3 and that alpha11beta1 regulates myofibroblast differentiation.

Inhibitory Role of Peroxisome Proliferator-Activated Receptor Gamma in Hepatocarcinogenesis in Mice and in Vitro

Although peroxisome proliferator-activated receptor gamma (PPARgamma) agonist have been shown to inhibit hepatocellular carcinoma (HCC) development, the role of PPARgamma in hepatocarcinogenesis remains unclear. We investigated the therapeutic efficacy of PPARgamma against HCC. PPARgamma-deficient (PPARgamma(+/-)) and wild-type (PPARgamma(+/+)) littermates were used in a diethylnitrosamine (DEN)-induced HCC model and treated with PPARgamma agonist (rosiglitazone) or the vehicle alone for 8 months. The effects of PPARgamma on HCC cell growth and apoptosis were examined using PPARgamma-expressing adenovirus (Ad-PPARgamma). PPARgamma(+/-) mice were more susceptible to DEN-induced HCC than PPARgamma(+/+) mice (94% versus 62%, P < 0.05), and rosiglitazone significantly reduced the incidence of HCC in PPARgamma(+/+) mice (vehicle 62% versus treatment 24%, P < 0.01), but not in PPARgamma(+/-) mice, indicating that PPARgamma suppresses hepatocellular carcinogenesis. A pronounced expression of PPARgamma was observed in a HCC cell line (Hep3B) infected with Ad-PPARgamma. Such induction markedly suppressed HCC cell viability (P < 0.01). Further, Hep3B infection with Ad-PPARgamma revealed a decreased proportion of cells in S-phase (12.92% versus 11.58%, P < 0.05), with arrest at G(2)/M phase (38.2% versus 55.68%, P < 0.001), and there was concomitant phosphorylation of the key G(2)/M phase inhibitors cdc25C and cdc2. PPARgamma overexpression increased cell apoptosis (21.47% versus 35.02%, P < 0.01), mediated by both extrinsic (Fas and tumor necrosis factor-alpha) and intrinsic (caspase-9, caspase-3, caspase-7, and poly[ADP-ribose] polymerase) pathways. Moreover, PPARgamma directly induced a putative tumor suppressor gene, growth differentiation factor-15. Loss of one PPARgamma allele is sufficient to enhance susceptibility to HCC. PPARgamma suppresses tumor cell growth through reducing cell proliferation and inducing G(2)/M phase arrest, apoptosis, and up-regulating growth differentiation factor-15. Thus, PPARgamma acts as a tumor-suppressor gene in the liver.

Oxidation of Cell Surface Thiol Groups by Contact Sensitizers Triggers the Maturation of Dendritic Cells

p38 mitogen-activated protein kinase (MAPK) has a crucial role in the maturation of dendritic cells (DCs) by sensitizers. Recently, it has been reported that the oxidation of cell surface thiols by an exogenous impermeant thiol oxidizer can phosphorylate p38 MAPK. In this study, we examined whether sensitizers oxidize cell surface thiols of monocyte-derived DCs (MoDCs). When cell surface thiols were quantified by flow cytometry using Alexa fluor maleimide, all the sensitizers that we examined decreased cell surface thiols on MoDCs. To examine the effects of decreased cell surface thiols by sensitizers on DC maturation, we analyzed the effects of an impermeant thiol oxidizer, o-phenanthroline copper complex (CuPhen). The treatment of MoDCs with CuPhen decreased cell surface thiols, phosphorylated p38 MAPK, and induced MoDC maturation, that is, the augmentation of CD83, CD86, HLA-DR, and IL-8 mRNA, as well as the downregulation of aquaporin-3 mRNA. The augmentation of CD86 was significantly suppressed when MoDCs were pretreated with N-acetyl-L-cystein or treated with SB203580. Finally, we showed that epicutaneous application of 2,4-dinitrochlorobenzene on mouse skin significantly decreased cell surface thiols of Langerhans cells in vivo. These data suggest that the oxidation of cell surface thiols has some role in triggering DC maturation by sensitizers.