Maria Luisa Colamussi

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-ζ translocation to the nucleus of NGF-treated PC12 cells

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738–29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non‐canonical 3‐phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC‐ζ nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3‐kinase (PI 3‐K) activityand its product, phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3‐K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3‐K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P3. Maximal translocation of PKC‐ζ from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG‐sensitive, PKC‐α. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1‐O‐octadeyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine, was unable to abrogate nuclear translocation of the DAG‐insensitive PKC‐ζ. These data suggest that a nuclear increase in PI 3‐K activity and PtdIns(3,4,5)P3 production are necessary for the subsequent nuclear translocation of PKC‐ζ. Furthermore, they point to the likelihood that PKC‐ζ is a putative nuclear downstream target of PI 3‐K during NGF‐promoted neural differentiation.—Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3‐kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC‐ζ translocation to the nucleus of NGF‐treated PC12 cells. FASEB J. 13, 2299–2310 (1999)

Stromal derived factor-1α (SDF-1α) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway

Stromal‐derived factor‐1α (SDF‐1α), the high‐affinity ligand of CXC‐chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF‐1α‐mediated Jurkat cell apoptosis was observed in serum‐free or serum‐containing cultures, peaked at SDF‐1α concentrations of 10–100 ng/ml, required 3 days to take place, and was completely blocked by the z‐VAD‐fmk tripeptide caspase inhibitor. Although SDF‐1α did not modify the expression of TNF‐α or that of TNF‐RI and TNF‐RII, it increased the expression of surface Fas/APO‐1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF‐1α to induce apoptosis was inhibited by an anti‐CD95 Fab′ neutralizing antibody. These findings suggest a role for SDF‐1α in the homeostatic control of CD4+ T‐cell survival/apoptosis mediated by the CD95‐CD95L pathway.

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides

The increase in human neutrophil cyclic adenosine monophosphate (cAMP) levels evoked by formylated peptides is significantly reduced in the presence of MDL 12330A, SQ 22536, GDPssS and clonidine, which inhibit the adenylyl cyclase system by acting at different sites in this enzyme complex. A similar effect is exerted by adenosine deaminase and dipyridamole, which alter the extracellular adenosine concentration. Neutrophil preincubation with adenylyl cyclase inhibitors or dipyridamole reduces chemotaxis and superoxide anion production triggered by peptides; adenosine deaminase, on the contrary, has no effect on neutrophil responses. Our results seem to indicate that: (1) the peptide-induced increase in neutrophil cAMP is due mainly to an action on the adenylyl cyclase system; (2) an enhancement of this cyclic nucleotide, even slight and necessarily transient, is required for chemotaxis and O2 production induced in neutrophils by formylated peptides; and (3) cAMP does not represent the crucial second messenger for adenosine in the modulation of neutrophil responses.

Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues

The N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-OMe (1) analogues for-Thp-Leu-Ain-OMe (2), for-Thp-Leu-Phe-OMe (3), for-Met-Leu-Ain-OMe (4), for-Met-Delta(z)Leu-Phe-OMe (5), for-Met-Lys-Phe-For-Met-Lys-Phe (6), for-Met-Leu-Pheol-COMe (7), and for-Nle-Leu-Phe-OMe (8) have been studied. Some of these have been found selective towards the activation of different biological responses of human neutrophils. In particular, peptides 2 and 3, which evoke only chemotaxis, are ineffective in enhancing inositol phosphate, as well as cyclic AMP (cAMP) levels. On the contrary, analogues 5 and 7, which induce superoxide anion production and degranulation, but not chemotaxis, significantly increase the levels of the two intracellular messengers, as is the case of the full agonists 1 and 6. The Ca(2+) ionophore A23187 also activates phospholipase C (PLC) and increases the nucleotide levels; when tested in combination with peptide 1 or 5, a supra-additive enhancement of cAMP concentration is obtained. The PLC blocker, U-73122, inhibits the formylpeptide-induced inositol phosphate formation, as well as cAMP increase. Moreover, this drug drastically reduces superoxide anion release triggered by 1 or 5, whereas it inhibits to a much lesser extent neutrophil chemotaxis induced by 1 or 2. Our results suggest that: (i) PLC stimulation is involved in cAMP enhancement by formylpeptides; (ii) the activation of PLC by formylpeptides, in conditions of increased Ca(2+) influx, induces a supra-additive enhancement of the nucleotide; (iii) the inability of pure chemoattractants to significantly alter the PLC activity or cAMP level, differently from full agonists or peptides specific in inducing superoxide anion release, appears as a general property. Thus, the activation of neutrophil PLC seems essential for superoxide anion release, but less involved in the chemotactic response.

Selective up-regulation of phospholipase C-β2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors

In this study, we have investigated the expression of phospholipase C‐β2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans‐retinoic acid (ATRA) by differentiating in a neutrophil‐like manner. We found that PLC‐β2, virtually absent in untreated NB4 cells, was strongly up‐regulated after ATRA‐induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC‐β2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC‐β2 expression also characterized the cytokine‐induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC‐β2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC‐β2 levels can predict the in vivo responsiveness to ATRA of APL patients.

MDL 12330A inhibits the non-neuronal adenylyl cyclase from the freshwater snail Planorbarius corneus, but the neuronal enzyme is activated by this compound

N-(Cis-2-phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL 12330A), considered an inhibitor of adenylyl cyclase, has been tested on the enzyme activity of neuronal and non-neuronal tissues from the freshwater snail Planorbarius corneus. The drug dose-dependently activates the basal as well as agonist-stimulated adenylyl cyclase in the ganglionic preparations, while it exerts an inhibitory effect on the enzyme present in the non-nervous tissues examined. SQ 22536 and forskolin, respectively an inhibitor and activator of adenylyl cyclase, behave as generally reported both in central and peripheral tissues of the snail. This is, to our knowledge, the first report of a stimulatory action of MDL 12330A on an adenylyl cyclase system.

Effect of serotonin and neuropeptides on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus

The effect of serotonin, FMRFamide and the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. The amine and the cardioactive peptide stimulated the enzyme, although with different potencies, in all tissues studied and, when tested in combination, an additive activation was obtained. FMRFamide induced differential effects in the various targets: marked stimulation of adenylate cyclase, additive to that provoked by serotonin or SCPB, in salivary glands; inhibition of the enzyme, both alone and in combination with the other neuromediators, in the nervous tissue; whereas no influence was found in adenylate cyclase activity in the buccal mass. In the last of these tissues, the peptide might act through an intracellular second messenger other than cyclic AMP. The responsiveness of adenylate cyclase to these neuromediators in all the central ganglia suggested that they can exert an important role as neurotransmitters and/or neuromodulators in the central nervous system of the snail. Moreover, in the light of the differential sensitivity of adenylate cyclase in the salivary glands and buccal mass, we suggest that serotonin, FMRFamide and SCPB modulate the feeding behaviour of P. corneus in a complex way.

Effects of Ca2+ and calmodulin on adenylyl cyclase activity in sheep olfactory epithelium.

Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca2+/calmodulin, as suggested by the results obtained both in Ca2+/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca2+ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca2+/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca2+/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed.