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PSL Quarterly Review, V. 51, N. 204 (1998): supplement

Moneta e Credito, V. 51, N. 201 (1998): supplemento

There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG 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 (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.

DOCK8-deficiency is an autosomal recessive primary immunodeficiency that is characterized by multiple abnormalities of the immune system, including a defect of NK cell cytotoxicity which could not be restored after IL2 stimulation. Nevertheless, unanswered questions remain regarding how the absence of DOCK8 leads to predisposition for malignancy, viral, fungal, and bacterial infections. To address these questions we have analyzed NK cell phenotype and functions in patients with DOCK8 deficiency. We observed that NK cells derived from five DOCK8-deficient patients displayed dramatically reduced cytotoxicity which was partially restored after IL-2 stimulation. Analysis of activating and inhibitory NK receptors, including KIRs molecules, chemokine receptors and activation markers on gated CD56+cells by cytofluorimetric analysis showed a substantial defect of CCR7 expression by CD56bright NK cells. Noteworthy, we have also detected the expression of NKG2C and of the chemokine receptor CXCR1 on CD56dull NK cells in DOCK8-deficient cells. Because CCR7 expression by NK cells can be induced after cell culture with IL18 we stimulated NK cells from DOCK-8 deficient patients with IL18. Despite unstimulated CD56bright NK cells from DOCK8 patients showed reduced CCR7 expression, we could not detect any increase of CCR7 on CD56 dull and bright NK cells of DOCK8-deficient patients, whereas CCR7 expression on NK cells derived from healthy donors significantly increased from 5% to 17%.Taken together our results suggest that NK cells of DOCK-8 deficient patients show reduced cytotoxicity and abnormal expression of the chemokine receptors CXCR1 and CCR7 suggesting an abnormal recruitment of these cells to secondary lymphoid organs., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

We have investigated the intranuclear distribution of High-mobility group proteins I/Y by means of immunofluorescent staining employing a variety of cell lines. Confocal scanning laser microscopy analysis revealed that High-mobility group proteins I/Y are present in an intranuclear fibrogranular network and mitotic chromosomes. In Caski, C4I, Flow 2002, and K562 cell lines, High-mobility group proteins I/Y were absent from nucleoli, whereas in HeLa cells they were present in this nuclear domain. Double immunolabeling studies showed that High-mobility group proteins I/Y co-localize with other key nuclear components such as NuMA, SC-35, and TAF(II)70. Nuclear reactivity for High-mobility group proteins I/Y dramatically decreased in apoptotic nuclei of HL-60 human leukemia cells. Our morphological data correlate well with previous biochemical and functional findings obtained by other investigators, who have demonstrated multiple roles for this class of polypeptides. However, they point to the likelihood that High-mobility group proteins I/Y are involved in as yet unidentified functions, most likely in the speckle domains of the nucleus. They also show that conceivably these proteins are also involved in apoptosis.

Several studies have demonstrated the existence of an autonomous intranuclear phosphoinositide cycle that involves the activation of nuclear PIPLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PIPLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-ß1. Nuclear PI-PLC-ß1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-ß1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-ß1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.

We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A.

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium- dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca (2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.