Paola Narducci

Nuclear apoptotic changes: An overview

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post‐translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components. J. Cell. Biochem. 82: 634–646, 2001.

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells.

The SK‐N‐MC neuroblastoma cell line, which expresses surface tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors TRAIL‐R2 and TRAIL‐R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short‐term (1–60 min) and long‐term (3–24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal‐regulated kinase (ERK), cAMP response element‐binding protein (CREB), nuclear factor kappa B (NF‐κB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c‐Jun NH2‐terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho‐CREB DNA binding activity in a phosphatidylinositol 3‐kinase (PI 3K)/Akt‐dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of κB (IκB)β and IκBε, but not IκBα, coupled to the nuclear translocation of NF‐κB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase‐8 and caspase‐3, and to induce apoptosis. Remarkably, caspase‐dependent cleavage of NF‐κB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase‐dependent apoptosis.

The nuclear matrix and apoptosis.

Abstract Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Abstract: Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation.

Constitutive Traffic of Melanocortin-4 Receptor in Neuro2A Cells and Immortalized Hypothalamic Neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds α-melanocyte-stimulating hormone (α-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of α-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toα-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that α-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Re-examination of the mechanisms regulating nuclear inositol lipid metabolism

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3‐kinase and phosphoinositide‐specific phospholipase C‐β1.

Insulin-like growth factor-I-dependent stimulation of nuclear phospholipase C-?1 activity in Swiss 3T3 cells requires an intact cytoskeleton and is paralleled by increased phosphorylation of the phospholipase

Swiss 3T3 mouse fibroblasts were exposed to 10 μM colchicine to disrupt microtubules, then stimulated with insulin‐like growth factor‐I. Immunoprecipitation experiments showed that insulin‐like growth factor‐I receptor and insulin receptor substrate‐1 were tyrosine phosphorylated to the same extent in both cells treated with colchicine and in those not exposed to the drug. Moreover, the activity of phosphatidylinositol 3‐kinase was not affected by incubation with colchicine. While in nuclei prepared from cells not exposed to colchicine it was possible to detect an insulin‐like growth factor‐I‐dependent increase in the mass of diacylglycerol, as well as stimulation of phospholipase C activity, no similar changes were observed in nuclei obtained from cells treated with colchicine. Activation of the nuclear phospholipase activity was paralleled by an increase of its phosphorylation. Immunofluorescent studies revealed that mitogen‐activated protein kinase did not translocate towards the nucleus when the cytoskeleton was depolymerized. These results show that in Swiss 3T3 cells some as yet unknown events necessary for the insulin‐like growth factor‐I‐dependent activation of nuclear polyphosphoinositide metabolism require the presence of an intact cytoskeleton and are situated down‐stream the activation of insulin receptor substrate‐1 and phosphatidylinositol 3‐kinase. Activation of nuclear phospholipase C‐β1 might be linked to its phosphorylation and translocation of mitogen‐activated protein kinase to the nucleus. J. Cell. Biochem. 72:339–348, 1999.

A quantitative study on the spatial and temporal ossification patterns of vertebral centra and neural arches and their relationship to the fetal age

A double-staining technique on 37 human embryos and fetuses (crown-rump length, CRL, between 38 and 116 mm) has been performed to study the ossification patterns of the vertebral column. Different growth sequences for centra and neural arches were observed. The survey of ossified centers suggested it was possible to relate significantly their appearance with the CRL. On the basis of already known data defining the developmental age in relationship to the latter parameter, we suggest their numerical evaluation as a further parameter for the assessment of the fetal age. Therefore, we have worked out a table that may be used either to determine the normal fetal growth, or when other parameters cannot be relied upon (i.e. in morphological diseases) for this aim.

Developmental pathways of vertebral centra and neural arches in human embryos and fetuses

SummaryThe ossification pathways of both vertebral centra (i.e., vertebral bodies) and neural arches were studied in human embryos and fetuses (CR-length between 38 and 116 mm). A clearing and double-staining method for whole embryo or fetus, using alcian blue and alizarin red S, allowed an easy and precise detection of the morphology of the whole vertebral column and every single vertebra. Both cartilaginous and bony components were clearly visible. Different temporal and topographical patterns of ossification were shown for the centra and arches; the latter were respectively proximaldistal (i.e., bidirectional from a defined starting tract in T10-L1) and cranial-caudal (i.e., monodirectional). The patterns could be related to the morphogenetic processes of other structures (i.e., muscles and nerves). Moreover, the numerical survey of ossification centers provided a possible parameter for the determination of the fetal developmental age. This could be useful in the study of pathological conditions.

Biochemical and morphological characterization of the nuclear matrix from apoptotic HL-60 cells.

We have characterized the nuclear matrix‐intermediate filament fraction from control and apoptotic HL‐60 cells. Apoptosis was induced by exposure to the topoisomerase I inhibitor, camptothecin. By means of two‐dimensional polyacrylamide gel electrophoresis, striking qualitative and quantitative differences were seen in the protein composition of the nuclear matrix–intermediate filament fraction obtained from apoptotic cells in comparison with controls. Western blotting analysis of apoptotic nuclear matrix proteins revealed degradation of some (topoisomerase IIα, SAF‐A) but not other (SATB1 and nucleolin) components. Moreover, immunofluorescent staining for typical matrix antigens (NuMA protein, lamin B, SC‐35) showed that in 35–40% of the structures prepared from apoptotic samples, marked changes in the subnuclear distribution of these proteins were present. Striking morphological differences between control and apoptotic samples were also detected at the ultrastructural level. These results demonstrate that both biochemical and morphological changes can be detected in the nuclear matrix prepared from apoptotic HL‐60 cells. J. Cell. Biochem. 72:35–46, 1999.