Non-stimulated Cells Research Articles

The Tyrosine Phosphatase SHP2 Regulates Focal Adhesion Kinase to Promote EGF-Induced Lamellipodia Persistence and Cell Migration

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of receptor tyrosine kinases (RTK) signaling. Furthermore, SHP2 is known to promote cell migration and invasiveness, key steps in cancer metastasis. To date, however, the mechanism by which SHP2 regulates cell movement is not fully understood. In the current report, a new role for SHP2 in regulating cell migration has been suggested. We show that SHP2 mediates lamellipodia persistence and cell polarity to promote directional cell migration in the MDA-MB231 and the MDA-MB468 basal-like and triple-negative breast cancer cell lines. We further show that SHP2 modulates the activity of focal adhesion kinase (FAK) by dephosphorylating pTyr397, the autophosphorylation site that primes FAK function. Because hyperactivation of FAK is known to counter the maturation of nascent focal complexes to focal adhesions, we propose that one of the mechanisms by which SHP2 promotes lamellipodia persistence is by downregulating FAK activity through dephosphorylation of pTyr397. The finding that inhibition of FAK activity partially restores EGF-induced lamellipodia persistence and cell migration in SHP2-silenced cells supports our proposition that SHP2 promotes growth factor-induced cell movement by acting, at least in part, on FAK. However, the effect of SHP2 inhibition in nonstimulated cells seems FAK independent as there was no significant difference between the control and the SHP2-silenced cells in pY397-FAK levels. Also, FAK inhibition did not rescue Golgi orientation defects in SHP2-silenced cells, suggesting that SHP2 acts through other mechanisms to promote cell polarity.

Kinetics of the CTLA-4 isoforms expression after T-lymphocyte activation and role of the promoter polymorphisms on CTLA-4 gene transcription

Cytotoxic T lymphocyte antigen 4 (CTLA-4) plays a key inhibitory role during T lymphocyte activation. The CTLA4 gene is translated into two proteic isoforms: a full-length protein (flCTLA-4) and a soluble counterpart. We explored the expression of both isoforms on healthy subjects. Whereas in non-stimulated cells the flCTLA-4 isoform is predominant, after stimulation the expression of the soluble form rapidly increases, reaching its maximum 24h after and falling again to the basal levels 72 h after stimulation. In contrast, the flCTLA-4 mRNA levels increase is slower, reaching the maximum level 72 h after stimulation. The presence of the T allele in the promoter positions -1722 and -318 is associated with an increased transcriptional activity and this effect seems to be synergic. We conclude that the kinetics of CTLA-4 isoform expression are sequential, and that the promoter polymorphisms -1722(C/T) and -318(C/T) are involved in the control of the CTLA4 transcription.

Cezanne Paints Inflammation by Regulating Ubiquitination

Hypoxia–reoxygenation can induce inflammation by activating nuclear factor (NF)-κB. In endothelial cells, this process is critical for the pathogenesis of many chronic inflammatory conditions, such as atherosclerosis and autoimmune disease. Recent publication from Evans’s laboratory shows the critical role of deubiquitinating enzyme Cezanne, regulating its extent of NF-κB activation and expression of inflammatory genes.1 In particular, they showed that the inhibition of polyubiquitination of TNF receptor associated factor (TRAF) 6 is a specific anti-inflammatory mechanism by Cezanne. In this editorial, we briefly review the TRAF6-mediated NF-κB signaling and other posttranslational modifications that play a key role in modulating endothelial cell inflammation. Article, see p 1583 NF-κB transcription factor complexes consist of a heterodimer of p65 (RelA) and p50 or p52.2 In most nonstimulated cells, p65-containing NF-κB complexes are kept in an inactive cytoplasmic form, bound to one family of inhibitor proteins, the inhibitory κBs (IκBs). Two IκB kinases, IKKα and IKKβ, target phosphorylation of IκB after hypoxia–reoxygenation, cytokine, or ultraviolet stress stimulation. Phosphorylation of IκBs promotes their ubiquitination and degradation by the proteasome, which releases the p65 complex, allowing it to translocate to the nucleus.3 An ubiquitin E2 conjugating enzyme of the ubiquitin-conjugating enzyme (Ubc)4/5 family and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box beta protein βTrCP) execute ubiquitination of IκB. Once IκB is phosphorylated, p-transducin repeat containing protein 1 (βTrCP1) and βTrCP2 associate with phosphorylated IκB.4,5 The polyubiquitinated IκB is selectively degraded by the 26S proteasome, and then mature p52 and p65 subunits translocate into the nucleus to increase NF-κB activity.4 It is now well established that NF-κB activation is a key event in inflammation-based cardiovascular pathogenesis, including atherosclerotic plaque formation, diabetes mellitus–mediated endothelial inflammation, and ischemia/reperfusion injury.6 The regulatory subunit NF-κB essential modulator (NEMO/IKKγ) of the cytoplasmic IκB …

The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: An ultrastructural, histochemical and immunocytochemical study

Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph.

Requirement of Vesicle-Associated Membrane Protein 721 and 722 for Sustained Growth during Immune Responses in Arabidopsis

Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle-associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.

Expression and activation of intracellular receptors TLR7, TLR8 and TLR9 in peripheral blood monocytes from HIV-infected patients.

TLR´s play a role in host defense in HIV infection recognizing the viral DNA or RNA. Their activation induces a signaling pathway that includes the proteins MyD88, IRAK4, TRAF6 and the transcription factor NF-kBp65. To determine the expression of TLR7, TLR8 and TLR9, and activation of its signaling pathway in monocytes from patients infected with HIV. Methods. Expression of TLR7, TLR8 and TLR9 was determined in monocytes from HIV-infected patients (n= 13) and control subjects (n= 13), which were activated with specific ligands. The expression of MyD88 and NF-kBp65 were determined by flow cytometry; IRAK4 and TRAF6 were studied by immunoblotting. No statistical difference was found in the expression of TLR7, 8 and 9 in monocytes from patients compared to controls, but we observed the non-significant increased expression of TLR9 in patients. The activation showed no significant difference in the expression of MyD88 and NF-kBp65 in patients when compared to controls, but were decreased in stimulated cells over non-stimulated cells. IRAK4 and TRAF6 were not detected. No statistical difference was observed in the expression of intracellular TLRs, MyD88 and NFkBp65 in monocytes from patients compared to controls. This was probably due to effective antiretroviral therapy being received at the time of study entry. Additional studies are needed under controlled conditions that include infected patients with and without ARVT, responders and non-responders, and work with different cell populations.

Reactivity of bovine WC1+ γδ T cells to viral dsRNA (P6107)

Abstract Bovine γδ T cells have properties of both innate and adaptive immune cells. However, detailed studies of how bovine γδ T cells execute their functions as innate or adaptive immune cells are lacking. In addition, their precise role in viral infections of cattle is not known. We have examined the reactivity of bovine γδ T cells to viral dsRNA isolated from bovine respiratory syncytial virus. Viral dsRNA was added to sorted peripheral blood WC1+ γδ T cells or directly delivered into the cells with DOTAP. Cell surface expression of CD80, CD335, MHC I and II, and synthesis of TNFα and IFNγ were investigated following stimulation with viral dsRNA. Addition of viral dsRNA to the culture of γδ T cells does not lead to changes in expression of CD335, MHC I and II but induces increased expression of CD80, although not statistically significant compared to non-stimulated cells. Direct delivery of viral dsRNA into WC1+ γδ T cells by DOTAP induced a twofold increase in expression of CD335 and MHC I with smaller increases in expression of MHC II and CD80. Although WC1+ γδ T cells synthesized low levels of TNFα and IFNγ in the presence of viral dsRNA or not, they significantly increased synthesis of IFNγ upon direct delivery of viral dsRNA into the cells. Results suggest that viral dsRNA may induce expression of molecules that promote innate function of γδ T cells and are discussed in context of pathways for enhancing bovine γδ T cell function against viral infections.

Abstract 3801: Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase .

Abstract Prostate cancer is the leading cause of cancer deaths in men here in the U.S. and over 240,000 new cases are predicted to be diagnosed this year. Initially, tumors arising in this gland rely on the androgen receptor (AR) and its ligand, dihydrotestosterone (DHT) for growth and survival. At this stage, androgen deprivation therapy (ADT) can lead to tumor regression. Unfortunately, patients typically relapse within two to three years and tumors no longer respond to ADT; this stage is termed castration-resistant prostate cancer (CRPC) for which there is no cure. At this stage, AR expression is either increased or mutationally activated and non-responsive to physiological levels of androgen. Several reports have shown that AR has both genomic signaling within nucleus and non-genomic signaling within the cytoplasm. Using prostate tumor cell line PC-3, expressing wild-type AR (PC3-AR), we reported that AR genomic signaling leads to an increase in integrin α6β1 transcription and expression, which in turn increases Bcl-xL expression, a mechanism that regulates prostate cancer cell survival. However, a role for AR and integrin α6β1 in prostate cancer metastasis has not been examined. In addition to promoting cell survival, integrins are critically important for cell movement and can activate Src. We observed that in the absence of androgen, PC3-AR cells have increased Src activity as well as enhanced cell migration and invasion relative to cells that do not expressing AR. Androgen responsive prostate cancer cell lines LNCaP, VCaP and C42 which express AR endogenously, have enhanced invasion and Src activity once stimulated with androgen. We found that knock-down of AR decreased Src activity in PC3-AR cells, as well as in LNCaP and C42 cells. We also found that inhibiting the expression of either AR or Src blocked the ability of cells to invade through matrix in vitro. However, neither AR nor Src possess the ability to degrade ECM proteins. Thus, we hypothesize that AR activates Src in a non-genomic fashion to promote a metastatic phenotype by the regulation of a downstream protease. To test our hypothesis, we set out to determine how AR/Src signaling controls the expression and/or activity of a protease that can degrade proteins in the ECM to promote metastasis. Our data shows that the protease downstream of AR/Src signaling is matriptase, a transmembrane serine protease that has been recently reported to cleave laminin. We observe matriptase cellular expression levels decrease in androgen responsive cell lines once stimulated with androgen as early as twenty minutes. This is suggestive that this is regulated through a non-genomic signaling mechanism. Also seen in these androgen stimulated cells within twenty minutes is that matriptase is shed and found within the media at higher levels relative to the media of non-stimulated cells. Citation Format: Jelani C. Zarif, Laura E. Lamb, Cindy K. Miranti. Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2013-3801

Deleterious Cholesterol Hydroperoxide Trafficking in Steroidogenic Acute Regulatory (StAR) Protein-expressing MA-10 Leydig Cells: IMPLICATIONS FOR OXIDATIVE STRESS-IMPAIRED STEROIDOGENESIS

Steroidogenic acute regulatory (StAR) proteins in steroidogenic cells are implicated in the delivery of cholesterol (Ch) from internal or external sources to mitochondria (Mito) for initiation of steroid hormone synthesis. In this study, we tested the hypothesis that under oxidative stress, StAR-mediated trafficking of redox-active cholesterol hydroperoxides (ChOOHs) can result in site-specific Mito damage and dysfunction. Steroidogenic stimulation of mouse MA-10 Leydig cells with dibutyryl-cAMP (Bt2cAMP) resulted in strong expression of StarD1 and StarD4 proteins over insignificant levels in nonstimulated controls. During incubation with the ChOOH 3β-hydroxycholest-5-ene-7α-hydroperoxide (7α-OOH) in liposomes, stimulated cells took up substantially more hydroperoxide in Mito than controls, with a resulting loss of membrane potential (ΔΨm) and ability to drive progesterone synthesis. 7α-OOH uptake and ΔΨm loss were greatly reduced by StarD1 knockdown, thus establishing the role of this protein in 7α-OOH delivery. Moreover, 7α-OOH was substantially more toxic to stimulated than nonstimulated cells, the former dying mainly by apoptosis and the latter dying by necrosis. Importantly, tert-butyl hydroperoxide, which is not a StAR protein ligand, was equally toxic to stimulated and nonstimulated cells. These findings support the notion that like Ch itself, 7α-OOH can be transported to/into Mito of steroidogenic cells by StAR proteins and therein induce free radical damage, which compromises steroid hormone synthesis.

Knockdown of CCR1 suppresses expression of Intercellular Cell Adhesion Molecule‐1 induced by hypoxia/reoxygenation in endothelial cells and reduces infarct sizes after myocardinal ischemia/reperfusion

It has been reported that CCL15, a CCR1 ligand, induces proatherogenic factors and has been implicated in atherosclerosis. The present study showed that exposure of human endothelial cells (ECs) to hypoxia and reoxygenation (H/R) resulted in a rapid increase in expression of ICAM‐1 and CCR1 ligands. Our results revealed that knockdown of CCR1 attenuated expression of ICAM‐1 in ECs after H/R. Stimulation of ECs with CCL15 increased expression of ICAM‐1 compared with those in non‐stimulated cells predominantly via CCR1. Our data showed that CCL15 increased cell adhesion of monocytoid cells to endothelial cells. Pretreatment of these cells with CCR1 siRNA prevented the CCL15‐induced expression of ICAM‐1 and monocyte adhesion to ECs. We further investigated expression of CCR1 ligands and ICAM‐1 in vivo after myocardinal ischemia and reperfusion (MIR). Enhanced expression of ICAM‐1 and CCR1 ligands has been demonstrated in rat hearts in vivo after MI‐R. Direct injection of CCR1 siRNA onto the ventricular wall reduced expression of ICAM‐1 in rat hearts activated by MI‐R. Staining of hearts revealed that silencing of CCR1 markedly reduced the infarct sizes after MI‐R. Our results indicate that CCR1 and its ligands play important roles in inflammatory responses after MI/R via upregulating expression of cell adhesion molecules.This work was supported by a grant from National Research Foundation of Korea.

311 SECRETED FACTORS FROM PERITUMOR ADIPOSE TISSUES OF CLEAR CELL RENAL CELL CARCINOMA INCREASED THE MOTILITY OF HUMAN CCRCC CELL LINE CAKI-2 VIA ENHANCEMENT OF WNT SIGNALING

You have accessJournal of UrologyKidney Cancer: Basic Research (II)1 Apr 2013311 SECRETED FACTORS FROM PERITUMOR ADIPOSE TISSUES OF CLEAR CELL RENAL CELL CARCINOMA INCREASED THE MOTILITY OF HUMAN CCRCC CELL LINE CAKI-2 VIA ENHANCEMENT OF WNT SIGNALING Achim Lusch, Vien Nguyen, Christopher Blair, Molly Baker, Victor Huynh, Xiaolin Zi, and Jaime Landman Achim LuschAchim Lusch Orange, CA More articles by this author , Vien NguyenVien Nguyen Orange, CA More articles by this author , Christopher BlairChristopher Blair Orange, CA More articles by this author , Molly BakerMolly Baker Orange, CA More articles by this author , Victor HuynhVictor Huynh Orange, CA More articles by this author , Xiaolin ZiXiaolin Zi Orange, CA More articles by this author , and Jaime LandmanJaime Landman Orange, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1695AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Perinephric adipose tissue (PAT) is metabolically active and manifests endocrine function. We evaluated the interaction between PAT and clear cell renal cell cancer (ccRRC) cells. METHODS PAT was collected from 49 patients (27 clear cell, 7 chromphobe, 1 papillary, 3 uncharacterized RCCs, and 11 benign pathology). PAT was harvested from patients undergoing renal surgery and cultured for 24 hours to generate conditioned media (CM). The effect of PAT CM on the proliferation and migration of ccRCC cell line Caki-2 was measured by MTT assay and Boyden chamber cell migration assay, respectively. In addition, Wnt/β-Catenin activity in PAT CM was examined as induced levels of β-Catenin. The proliferative and migratory responses are expressed as a stimulation index (SI) calculated by dividing the mean number of proliferative or migratory cells of PAT CM-stimulated wells by those of non-stimulated wells. Wnt response is similarly expressed as a ratio of mean Wnt activity of PAT CM-stimulated cells divided by that of non-stimulated cells. RESULTS PAT CMs from pT3 ccRCC patients resulted in a significant increase of CaKi-2 cell migration (mean SI and standard deviation, 1.80±0.52, P<0.05) compared to those at pT1 ccRCC (1.36±0.71, P>0.05) and pT2 (1.60±1.22, P>0.05), respectively. Conversely, the mean proliferative SIs of PAT CMs from ccRCC patients decreased as clinical stage advanced (pT1 versus pT3, 1.28± 0.37 vs. 0.83± 0.08). PAT CMs from patients with benign pathology has no significant effect on proliferation and migration. PAT CMs from chromophobe RCC also significantly increased the migration of Caki-2 (migratory SI, 1.96 ± 1.01) with no effect on proliferation (SI, 1.02 ± 0.50). High Fuhrman Grade is associated with increased migration by PAT CMs. Increased tumor sizes were inversely associated with proliferative SIs (correlation coefficient, -0.45). The increased migration of Caki-2 cells is associated with enhanced Wnt activity in PAT CMs. CONCLUSIONS We demonstrate a novel interaction between PAT and RCC which may facilitate understanding of cancer cell migration and metastasis in RCC patients. Further analysis is in progress. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e126 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Achim Lusch Orange, CA More articles by this author Vien Nguyen Orange, CA More articles by this author Christopher Blair Orange, CA More articles by this author Molly Baker Orange, CA More articles by this author Victor Huynh Orange, CA More articles by this author Xiaolin Zi Orange, CA More articles by this author Jaime Landman Orange, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Membrane Microdomains and Cytoskeleton Organization Shape and Regulate the IL-7 Receptor Signalosome in Human CD4 T-cells

Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.

Implementing Enzyme-Linked Immunosorbent Assays on a Microfluidic Chip To Quantify Intracellular Molecules in Single Cells

Cell-to-cell differences play a key role in the ability of cell populations to adapt and evolve, and they are considered to impact the development of several diseases. Recent advances in microsystem technology provide promising solutions for single-cell studies. However, the quantitative chemical analysis of single-cell lysates remains difficult. Here, we combine a microfluidic device with the analytical strength of enzyme-linked immunosorbent assays (ELISA) for single-cell studies to reliably identify intracellular proteins, secondary messengers, or metabolites. The microfluidic device allows parallel single-cell trapping and isolation in 625-pL microchambers, repeated treatment and washing steps, subsequent lysis and analysis by ELISA. Using a sandwich ELISA, we quantitatively determined the concentration of the enzyme GAPDH in single U937 cells and HEK 293 cells, and found amounts within a range of a few (1-4) attomol per cell. Furthermore, a competitive ELISA is performed to determine the concentration of the secondary messenger cyclic adenosine monophosphate (cAMP) in MLT cells, in response to the hormone lutropin. We found the half maximal effective concentration (EC50) of lutropin to have an average value of 2.51 ± 0.44 ng/mL. Surprisingly, there were large cell-to-cell variations for all supplied lutropin concentrations, ranging from 36 to 536 attomol cAMP for nonstimulated cells and from 80 to 1040 attomol cAMP for a concentration around the EC50 (3 ng/mL). Because of the high sensitivity and specificity of ELISA and the large number of antibodies available, we believe that our device provides a new, powerful means for single-cell proteomics and metabolomics.

Protein Phosphatase 2A Catalytic Subunit α Plays a MyD88-Dependent, Central Role in the Gene-Specific Regulation of Endotoxin Tolerance

MyD88, the intracellular adaptor of most TLRs, mediates either proinflammatory or immunosuppressive signaling that contributes to chronic inflammation-associated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of the MyD88 interactome, we found that in endotoxin-tolerant macrophages, protein phosphatase 2A catalytic subunit α (PP2Ac) enhances its association with MyD88 and is constitutively activated. Knockdown of PP2Ac prevents suppression of proinflammatory genes and resistance to apoptosis. Through site-specific dephosphorylation, constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex and broadly suppresses the activities of multiple proinflammatory/proapoptotic pathways as well, shifting proinflammatory MyD88 signaling to a prosurvival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence proinflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions.

Cytokine production by non-stimulated peripheral blood NK cells and lymphocytes in early-onset severe pre-eclampsia without HELLP

Preeclampsia involves an exacerbated maternal inflammatory response that suggests a possible role of innate immunity. NK cells can promote this kind of response through cytokine production and the expression of activating or inhibitory receptors. The aims of the present study were to explore cytokine production by peripheral blood mononuclear cells, as well as cytotoxic ability and receptor expression for HLA-E and HLA-G molecules in peripheral natural killer (NK) cells of women with early-onset severe preeclampsia without HELLP (hemolysis, elevated liver enzyme levels and a low platelet count) syndrome. The expression of the ILT2, KIRDL4, NKG2A, and NKG2C receptors and of cytotoxic activity was measured in non-stimulated NK cells, whereas the intracellular expression of IL-4, IL-10, IL-13, IL-12, IFNγ, TNF and VEGF, was assessed in non-stimulated peripheral blood mononuclear cells subsets using flow cytometry. Circulating soluble HLA-G was also determined by ELISA. The intracellular cytokines tested were significantly higher in NK cell subsets from severely preeclamptic women compared with the control group. On the other hand, the percentage of NK cells expressing NKG2A or NKG2C and the cytotoxic activity of NK cells were significantly higher in severely preeclamptic women. Furthermore, there was a significant correlation between urine protein concentration and soluble human leukocyte antigen G (soluble HLA-G) in serum. We conclude that patients with early-onset severe preeclampsia without HELLP syndrome have increased NK cell function related to cytokine production, cytotoxicity and expression of lectin-like receptors such as NKG2.

Adiponectin Receptors Form Homomers and Heteromers Exhibiting Distinct Ligand Binding and Intracellular Signaling Properties

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

Differential effects of the persistent DDT metabolite methylsulfonyl-DDE in nonstimulated and LH-stimulated neonatal porcine Leydig cells

3-Methylsulfonyl-DDE (MeSO2-DDE) is a potent adrenal toxicant formed from the persistent insecticide DDT. MeSO2-DDE is widely present in human plasma, milk and fat, and in tissues of marine mammals. In the present study, we investigated endocrine-disrupting properties of MeSO2-DDE in primary neonatal porcine Leydig cells. Unstimulated and LH-stimulated cells were exposed to MeSO2-DDE at concentrations ranging from 0.6 to 20μM for 48h. Cell viability, hormone secretion and expression of steroidogenesis related genes were recorded. Secretion of testosterone and estradiol was increased in a concentration-dependent fashion in unstimulated Leydig cells, while in LH-stimulated cells, secretion of testosterone, estradiol and progesterone was decreased. The expression of important steroidogenic genes was down-regulated both in unstimulated and LH-stimulated cells. Notably, no significant impairment of cell viability occurred at any exposure except the highest concentration (20μM) in LH-stimulated cells. This indicated that the effects on hormone secretion and gene expression were not caused by cytotoxicity. We conclude that the adrenal toxicant MeSO2-DDE disrupts hormone secretion in a complex fashion in neonatal porcine Leydig cells. The different endocrine responses in unstimulated and LH-stimulated cells imply that the endocrine disruptive activity of MeSO2-DDE is determined by the physiological status of the Leydig cells.

The Influence of Toll-Like Receptor Stimulation on Expression of EBV Lytic Genes

Epstein-Barr virus (EBV) establishes latency in the resting memory B-cell compartment. It has been recently suggested that maintenance of chronic infection is dependent on periodic reactivation. Although the stimuli for EBV reactivation in vivo during natural infections are largely unknown, there is evidence indicating that heterologous infections could trigger herpesviruses reactivation. The purpose of this work was to identify the influence of Toll-like receptors stimulation on EBV replication in EBV latently infected Burkitt lymphoma cells (P3HR-1, Raji and Namalwa). The cells were stimulated with Pam3CSK4 (synthetic triacylated lipoprotein), PolyI:C (synthetic analog of dsRNA), LPS (lipopolysaccharide from E.coli), measles virus (MeV) and PMA (phorbol myristate acetate). Non-stimulated cells (NS) served as control. EBV expression was investigated at mRNA level for three viral lytic genes: BZLF1 (immediate early, ZEBRA), BALF2 (early, EA) and BcLF1 (late, VCA). Additionally, the effect of stimulation on NF-kBp65 and inflammatory cytokines (IL-lb, IL-6, IL-8, IL-10, IL-12p70, and TNF) was investigated. Stimulation of TLRs led to limited changes in EBV expression manifesting as increase of ZEBRA at mRNA level in cells treated with PolyI:C and Pam3CSK4. Stimulation with PolyI:C, Pam3CSK4 and LPS also lead to considerable increase of NF-kBp65, while increased levels of inflammatory cytokines were observed for IL-8, TNF and IL-6 in cells treated with PMA and MeV. In conclusion, the results of our experiments support the suggestion that TLRs stimulation with microbial ligands influences EBV virus replication.

Role of Mitochondrial NCX on CXCL12-Induced Chemotaxis in A20 B Lymphocytes

Lymphocyte chemotaxis plays important roles in immunological reactions. It has been reported that mitochondria redistribute at the uropod during T lymphocyte migration triggered by a chemokine ligand (CXCL12). However, how mitochondria are involved in lymphocyte chemotaxis has been unclear. We studied roles of mitochondria Ca2+ handling proteins, Na-Ca exchange (NCXm) and Ca2+ uniporter, on CXCL12-induced chemotaxis in A20 B lymphocytes. CXCL12 (100 ng/ml) increased transwell migration of A20 cells from 4.6 ± 0.5 % (non-stimulated cells) to 12.6 ± 0.6 % (P < 0.05). This increase was dose-dependently inhibited by CGP-37157 (an inhibitor of NCXm), but not affected by Ru360 (an inhibitor of mitochondrial Ca2+ uniporter). Knock-down of a gene of NCXm (NCLX) by siRNA reduced NCLX protein expression to 52 ± 9 % and inhibited transwell migration similarly to CGP-37157. In the 8 hrs observation of cell migration under microscope, mean displacement of NCLX siRNA cells without CXCL12 was larger (21.4 ± 1.5 μm) than control siRNA cells (13.5 ± 2.2 μm, P < 0.05), and applying CXCL12 did not increase mean displacement and percentage of directional migration in NCLX siRNA cells. Intracellular Ca2+ measured by fura-2 was higher in NCLX siRNA cells (fura-2 ratio 0.49 ± 0.01) than the control siRNA cells (0.45 ± 0.01, P < 0.05) in the absence of CXCL12. After 2 hrs CXCL12 stimulation, the intracellular Ca2+ increased in control siRNA but not in NCLX siRNA cells. Treating A20 cells with BAPTA-AM (an intracellular Ca2+ chelator) suppressed the cell migration. Our results suggest that NCLX-mediated Ca2+ signaling is associated with CXCL12-induced chemotaxis in B lymphocytes.

IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity.

Event Abstract Back to Event IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity. Texia T. Riquelme1, L.Pablo Cid1, Francisco V. Sepulveda1 and Carlos A. Flores1* 1 Centro de Estudios Científicos, Chile Cystic fibrosis (CF) primary dysfunction in epithelial transport concurs with deregulation of the immune system in human patients and animal models. We observed that when the KCNN4 channel is genetically silenced in the CF mouse lethality is drastically reduced with no improvement of intestinal function. The KCNN4 channel is broadly expressed including the immune system. We aim to test if KCNN4 channel inhibition is involved in immune system dysfunctions associated with CF. CF animals presented a 4-fold increase of intestinal mast cells, the value obtained in the KCNN4-null/CF mice was lower and comparable to controls. IgE-induced migration was 2-fold increased in control and CF mast cells compared with non-stimulated cells. Migration was blocked when the KCNN4 specific inhibitor TRAM-34 was used. Migration was completely impaired in the KCNN4-null and KCNN4-null/CF cells. Serum IgE level was increased in the CF as in the KCNN4-null/CF mice. Inhibition of KCNN4 blocks migration in control and CF mast cells. Hyperplasia of mast cells in the CF mice is localized in the intestinal tissue and can be due to increased migration induced by the higher levels of IgE. Mast cell reduction on intestine of KCNN4-null/CF mice is due to the impairment in migration of mast cells and not to reduced IgE levels. The origin of IgE in the CF mice remains a matter of investigation. KCNN4 inhibtion can be a useful pharmacological target on inflammatory CF disease where mast cells can act as enhancers of inflammation. Acknowledgements FONDECYT 111408 and CONICYT-BFP Keywords: IgE, Mast Cells, Kcnn4, Cystic Fibrosis, mice models Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Riquelme TT, Cid L, Sepulveda FV and Flores CA (2013). IgE-dependent mast cell hyperplasya in the intestine of cystic fibrosis mouse model needs KCNN4 channel activity.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01071 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Carlos A Flores, Centro de Estudios Científicos, Valdivia, Los Rios, Chile, cflores@cecs.cl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Google Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Google Scholar Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores PubMed Texia T Riquelme L.Pablo Cid Francisco V Sepulveda Carlos A Flores Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.