T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Cell cycle regulation of mitochondrial function.

Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response.

Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.

OBJECTIVES: Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics. MATERIALS AND METHODS: Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC. RESULTS: All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively. CONCLUSIONS: 5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.

N-WASP is required for B-cell-mediated autoimmunity in Wiskott-Aldrich syndrome.

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS.

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

System-level characterization of Th2 cell development and immune cell responses to ZnO and TiO2 nanoparticles

Asthma and allergy are common diseases and their prevalence is increasing. One of the hypotheses that explains this trend is exposure to inhalable chemicals such as traffi c-related air pollution. Epidemiological research supports this theory, as a correlation between environmental chemicals and allergic respiratory diseases has been found. In addition to ambient airborne particles, one may be exposed to engineered nanosized materials that are actively produced due to their favorable physico-chemical properties compared to their bulk size counterparts. On the cellular level, improper activity of T helper (Th) cells has been connected to allergic reactions. Th cells can differentiate into functionally different effector subsets, which are identifi ed according to their characteristic cytokine profi les resulting in specifi c ability to communicate with other cells. Th2 cells activate humoral immunity and stimulate eradication of extracellular pathogens. However, persistent predominance of Th2 cells is involved in a development of number of allergic diseases. The cytokine environment at the time of antigen recognition is the major factor determining the polarization of a naïve Th cell. Th2 cell differentiation is initiated by IL4, which signals via transcription factor STAT6. Although the importance of this pathway has been evaluated in the mouse studies, the signaling components involved have been largely unknown. The aim of this thesis was to identify molecules, which are under the control of IL4 and STAT6 in Th cells. This was done by using system-level analysis of STAT6 target genes at genome, mRNA and protein level resulting in identifi cation of various genes previously not connected to Th2 cell phenotype acquisition. In the study, STAT6-mediated primary and secondary target genes were dissection from each other and a detailed transcriptional kinetics of Th2 cell polarization of naïve human CD4+ T cells was collected. Integration of these data revealed the hierarchy of molecular events that mediates the differentiation towards Th2 cell phenotype. In addition, the results highlighted the importance of exploiting proteomics tools to complement the studies on STAT6 target genes identifi ed through transcriptional profi ling. In the last subproject, the effects of the exposure with ZnO and TiO2 nanoparticles was analyzed in Jurkat T cell line and in primary human monocyte-derived macrophages and dendritic cells to evaluate their toxicity and potential to cause infl ammation. Identifi cation of ZnO-derived gene expression showed that the same nanoparticles may elicit markedly distinctive responses in different cell types, thus underscoring the need for unbiased profi ling of target genes and pathways affected. The results gave additional proof that the cellular response to nanosized ZnO is due to leached Zn2+ ions. The approach used in ZnO and TiO2 nanoparticle study demonstrated the value of assessing nanoparticle responses through a toxicogenomics approach. The increased knowledge of Th2 cell signaling will hopefully reveal new therapeutic nodes and eventually improve our possibilities to prevent and tackle allergic infl ammatory diseases.

Cellular signaling with nitric oxide and cyclic GMP

During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

TRP channels, omega-3 fatty acids, and oxidative stress in neurodegeneration: from the cell membrane to intracellular cross-links

The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1(p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.

Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.

Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.