COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region

Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine

<p>Abstract</p> <p>Background-</p> <p>Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission.</p> <p>Results-</p> <p>In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice.</p> <p>Conclusions-</p> <p>Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.</p

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

BACKGROUND: Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. METHODOLOGY: The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. mu opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. PRINCIPAL FINDINGS: We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca(2+). LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca(2+). LPS treatment increased mu opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. CONCLUSIONS: Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils

A PKC-Dependent Recruitment of MMP-2 Controls Semaphorin-3A Growth-Promoting Effect in Cortical Dendrites

There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3). Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaMbinding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems

Rôle du pH intracellulaire dans le contrôle de la différenciation des cellules oligodendrocytaires

Une augmentation spontanée du pH intracellulaire (pHi) des précurseurs oligodendrocytaires (OLP) est enregistrée au début de leur différenciation. Sur divers types cellulaires, des changements similaires de pHi constituent souvent une réponse à l'application de divers facteurs (facteurs de croissance, hormones). Nous avons analysé le rôle du pHi dans le déclenchement de la différenciation et dans les processus d'élaboration des prolongements des OLP. Les effets sur le pHi d'un agent pro-différenciant (l'acide rétinoïque, AR) et d'une molécule de guidage contrôlant la morphologie des oligodendrocytes (la sémaphorine 3A, Sema3A), ont été analysés par microfluorimétrie du BCECF. Les effets de manipulations expérimentales du pHi sur la morphologie des OLP ont été quantifiés par une méthode originale d'analyse basée sur l'utilisation de la géométrie fractale. L'application d'AR stimule la différenciation des OLP, un doublement du nombre de cellules exprimant le galactocérébroside est observé après 72 heures de traitement. Parallèlement, l'addition d'AR entraîne une augmentation du pHi de 0,25 unité. Cette alcalinisation est directement liée à l'augmentation d'activité de l'échangeur Na+/H+. La suppression expérimentale de cette alcalinisation empêche la stimulation de la différenciation par l'AR. Inversement, la reproduction expérimentale de cette alcalinisation, en l'absence d'AR, stimule la différenciation dans les mêmes proportions que l'AR. Nous avons également montré que cette alcalinisation induisait une activation des MAPK ERK1/2. L'utilisation d'autres valeurs expérimentales de pHi a permis de montrer que le taux de différenciation des OLP est principalement dépendant de pHi. Nous avons montré que les OLP expriment de manière fonctionnelle la plexine-A1, une sous unité du récepteur de la Sema3A. L'addition de Sema3A induit une diminution importante de la complexité morphologique des OLP. Parallèlement, Sema3A induit une acidification intracellulaire transitoire de 0,13 unité pH, concomitante d'une diminution d'activité des MAPK ERK1/2. La suppression expérimentale de l'acidification observée inhibe totalement l'effet de Sema3A sur la morphologie des OLP. Ces résultats démontrent que le pH intracellulaire constitue l'un des signaux impliqués dans les processus de différenciation et de croissance cellulaires.A spontaneous increase of intracellular pH (pHi) is observed at the beginning of oligodendrocytes precursors (OLP) differentiation. On many cell types, such pHi changes are induce by multiple factors (growth factors, hormones). We have analyzed the role of such pHi changes during OLP differentiation and processes outgrowth. The effects on pHi of retinoïc acid (RA), that promotes OLP differentiation, and of semaphorin 3A (Sema3A), that inhibits processes outgrowth, were measured by microfluorimetric analysis of the pH-sensitive probe BCECF. Effects of experimental pHi changes on OLP morphology were analyzed by using the fractal geometry. After 72 hours, application of RA promotes differentiation : a 2-fold increase in the number of cells expressing the galactocerebroside is observed. This effect was accompanied by an intracellular alkalinization of 0.25 pH unit. This alkalinization is due to an increase in the activity of the Na+/H+ exchanger. Blocking this alkalinization inhibits RA-stimulated differentiation. Conversely, mimicking this alkalinization in the absence of RA, induce OLP to differentiate. By analyzing different conditions, we demonstrate that OLP differentiation is dependent on pHi. Furthermore, an intracellular alkalinization from 6.88 to 7.13 is able to induce MAPK ERK1/2 activation. Oligodendrocytes express plexine-A1, one of the component of the Sema3A receptor. Addition of Sema3A greatly inhibits oligodendrocyte morphological complexity. Sema3A also induce an intracellular acidification of 0.13 pH unit, that is concomitant with a decrease in ERK1/2 activity. The inhibitory effect of Sema3A is counteracted by preventing this intracellular acidification. These results demonstrated that intracellular pH is a signal controlling OLP differentiation and processes outgrowt.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF