Nuclear Phosphoinositide Research Articles

Topology of inositol lipid signal transduction in the nucleus.

An increasing body of evidence shows that many of the key inositol lipids and enzymes responsible for their metabolism reside in nuclei. Moreover, the association of the nuclear phosphoinositide cycle with progression through the cell cycle and commitment toward differentiation has built a wider picture of the implications of phosphoinositides in the control of nuclear functions. This article reviews a central aspect of inositide nuclear signaling, i.e., the spatial organization of the signaling system within the nucleus in relationship to the nuclear organization in functional domains. Most of the evidence obtained with a variety of confocal and electron microscopy immunocytochemical techniques indicates that the phosphoinositides, the enzymes required for their synthesis and hydrolysis, and the targets of the lipid second messengers are localized at ribonucleoprotein structures involved in the transcript processing in the interchromatin domains. These findings demonstrate that nuclear inositol lipids exist in a nonmembranous form, linked to structural nuclear proteins of the inner nuclear matrix. They also suggest that the inositol signaling in the nucleus is completely independent of that at the cell surface and that it probably preceded in evolution the systems that are present at the cytoskeletal and cell membrane level.

Nuclei contain two differentially regulated pools of diacylglycerol

A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1,2] that is regulated during progression through the cell cycle [1,3], differentiation [1,2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1,3,4], which is important in the activation of nuclear protein kinase C (PKC) [5–7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3,8–11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cβ1 (PIC-β1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs).

Nuclear calcium release by InsP3-receptor channels plays a role in meiosis reinitiation in the mouse oocyte

Our purpose was to investigate the presence of nuclear specific elements of the phosphoinositide pathway, and the link between nuclear calcium events and the first step of meiosis resumption, i.e. germinal vesicle breakdown (GVB) in mouse immature oocytes. Using confocal laser scanning microscopy, we analyzed the effects of nuclear microinjection of inositol 1,4,5-trisphosphate (InsP3), heparin and anti-InsP3 receptor monoclonal antibodies on both spontaneous nuclear and cytoplasmic calcium oscillations, as well as the effects of these components on the GVB. First we observed that nuclear Ca 2+ events were dependent upon both nucleoplasmic and cytoplasmic InsP3 levels, highlighting a cross-talk between the GV and the cytoplasm concerning the Ca 2+/InsP3 pathway. We demonstrated also that: 1) type 1 InsP3 receptors were localized at the nuclear membrane level while type 3 were absent from the nucleus; 2) calcium release from nuclear stores was mediated by type 1 rather than type 3 InsP3 receptor associated channels; 3) the anti-InsP3 R-1 mAB microinjected into the nucleus inhibited the GVB. These results demonstrate that reinitiation of meiosis requires an increase in nuclear phosphoinositide dependent Ca 2+. Thus, the role of nuclear Ca 2+ homeostasis is discussed with particular emphasis on nuclear envelope dynamics.

The germinal vesicle of the mouse oocyte contains elements of the phosphoinositide cycle: what is their role at meiosis resumption?

The role of the nuclear phosphoinositide (PI) cycle during meiotic resumption in mouse oocytes was examined. First, using indirect immunofluorescence staining with specific monoclonal antibodies (mAbs) against elements of this cycle, the presence of inositol trisphosphate receptors (IP3Rs) (IP3R-1 or IP3R-3) or phosphoinositide-phospholipase (PLC) isoforms (PLC beta 1 or PLC gamma 1) was monitored in the germinal vesicle (GV). Using confocal laser scanning microscopy, we analysed the effects of the nuclear microinjection of these antibodies on both spontaneous nuclear calcium oscillations and meiosis resumption. Immunostainings showed that IP3R-1 and PLC beta 1 isoforms were both present in the GV, whereas IP3R-3 and PLC gamma 1 isoforms were not. The anti-IP3R-1 mAbs or the anti-PLC beta 1 mAbs microinjected into the GV, induced inhibition of both the nuclear Ca2+ oscillations and the meiotic process, whereas the anti-IP3R-3 mAbs and the anti-PLC gamma 1 mAbs did not. We concluded that a specific nuclear PI cycle is present in the mouse oocyte and meiosis resumption requires a specific nuclear phosphoinositide-dependent Ca2+ signal.

Control of Expression of PLCβ1by LAC Repressor System: Relationship between Nuclear PLCβ1Overexpression and Growth Factor Stimulation

Swiss 3T3 cells have a nuclear phosphoinositide signalling system which is under the control of insulin-like growth factor I (IGF-I) and acts separately from that at the plasma membrane. By using the Lac repressor system we were able both to obtain the inducible overexpression of phospholipase C β1(PLC β1) and to determine its subcellular localisation and partitioning. Moreover, by comparing the level of expression at the nucleus and the percentage of cells actively incorporating bromodeoxyuridine (BrdU) in S phase it has strengthened the issue of the importance of this PLC in the onset of DNA synthesis mediated by IGF-I. In addition, this system appears to be a very powerful tool for further analysis of the downstream events following the activation of nuclear PLC β1.

Intranuclear Translocation of Phospholipase C β2 during HL-60 Myeloid Differentiation

Phospholipases C (PLC) β3, γ1, and γ2 were detected in nuclei of HL-60 promyelocitic leukaemia cells. When HL-60 cells undergo terminal myeloid differentiation in the presence of ATRA, the β2 isoform appeared inside nuclei and was up-regulated until 72 hours of ATRA treatment. The β3 isozyme was also increased until 72 hours and both isoforms lowered their intranuclear amount at 96 hours and following days of treatment. By contrast PLC γ1 and γ2 progressively increased in the nucleus during granulocytic differentiation even after 72 hours of treatment. Terminal differentiation was characterised by the expression of high levels of PLC γ1 and γ2 and by low levels of PLC β2 and β3 in the nucleus. PIP2and PIP hydrolysis paralleled the prevalence of the β or γ subfamily, respectively. Moreover, at all the examined times no changes of PLCs in the whole cell were detectable, indicating ade novonuclear translocation of the β2 and an increased accumulation of β3, γ1, and γ2 isoforms. Thus, the intranuclear presence, expression, and activity of PLC isozymes, which are modulated during differentiation of HL-60 cells, implicate a role for nuclear phosphoinositide signalling in the process of cell maturation. In particular the nuclear translocation of PLC β2 candidates this PLC as a key enzyme in the granulocytic differentiative commitment of HL-60 cells.

Phosphoinositide Signalling in Nuclei of Friend Cells: DMSO-Induced Differentiation Reduces the Association of Phosphatidylinositol-Transfer Protein with the Nucleus

Friend erythroleukemia cells have a nuclear phosphoinositide cycle which is related to both mitogen-stimulated cell growth and erythorid differentiation. Because of the important role of the phosphatidylinositol-transfer protein (PI-TP) in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis, we have analysed nuclei isolated from Friend cells for the presence of PI-TP. By Western Blotting it was demonstrated that both intact nuclei and nuclei deprived of the outer membrane contained the PI-TPα isoform. Upon induction of erythroid differentiation by DMSO, the amount of nuclear PI-TPα was greatly diminished. As shown previously, under these same conditions, nuclear phospholipase Cβ1(PLCβ1) is down-regulated as well.

Stimulation of Nuclear Polyphosphoinositide Synthesis by GTP-γ-S: A Potential Regulatory Role for Nuclear GTP-Binding Proteins

The nonhydrolyzable GTP analogue GTP-γ-S was capable of stimulating in vitro phosphorylation of polyphosphoinositides in isolated nuclei prepared from mouse erythroleukemia cells. On the contrary, GDP-β-S was ineffective. The stimulation was not detectable when nuclei were prepared from erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide. Both nuclear phosphomonoesterase and phospholipase C activities were not influenced by GTP-γ-S. Our results point to the likelihood that nuclear phosphoinositide kinases might be regulated by a GTP-binding protein.

Nuclear Ca(2+)-fluxes and phosphoinositides in plants.

Complex signalling events are involved in the conversion of primary extracellular signals into intracellular responses. Two signal-induced second messengers have been at the focus of attention for more than a decade; these are CaZ+ and inositol( 1,4,5)trisphosphate [ 11. Many important functions for these messengers have been identified in mammalian cells, and the possibility that Ca*+ and Ins(1,4,5)P3 also have specific roles to play in nuclear signalling events has become apparent. The nucleus in mammalian cells has, unexpectedly, been found to possess a phosphoinositide system distinct from the more familiar plasma membrane phosphoinositide system [see ref 2 for review]. Although there is still some debate about the exact location of the nuclear phosphoinositide system it appears that several of the important functional components are localized inside the nucleus rather than in the nuclear membrane($ [2]. It has further been shown that the nuclear phosphoinositide system, in several cell types, is likely to be under the control of receptors localized in the plasma membrane [3]. The nucleus in mammalian cells has been found to contain an active endoplasmic reticulum (ER)-type Caz+ pump [4] and Ins(1,4,5)P3 has been shown to be able to release Caz+ from isolated nuclear preparations [5]. Many similarities exist between signalling systems in mammalian cells and plants and both Caz+fluxes and a plant phosphoinositide system have been shown to be involved in cell signalling events [6]. To investigate the possibility that Caz+-fluxes and phosphoinositide turnover may occur in plant nuclei we have developed a novel detergent-free method for the isolation of intact nuclei from carrot (Doueus carota) protoplasts. The method relies on gentle sonication to break open the plasma membrane and the released nuclei are further purified by gradient centrifugation. The apparent purity of nuclei is high, and using a number of different criteria we have found that the nuclear membranes remain largely intact. Figure 1 shows a carrot cell nucleus stained with the membrane-specific dye DiOC,.

Identification of PI-PLC β1, γ1, and δ1 in rat liver: Subcellular distribution and relationship to inositol lipid nuclear signalling

The subcellular distribution of PI-PLC β1, γ1, and δ1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC β1 is predominantly nuclear, while γ1 is largely cytoplasmic and δ1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms β1 and γ1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-β1 and -γ1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC β1 and γ1 play a central role in modulating the nuclear phosphoinositide cycle.

A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.

Nuclear inositol lipid cycle and differentiation

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform β1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both β1 and -γ1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC β1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC β1 message, whilst the amount of PLC γ1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC β1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

The nuclear phosphoinositide cycle — does it play a role in nuclear Ca 2+ homoeostasis?

The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca 2+ changes changing in response to cytosolic Ca 2+, with little evidence for an independently controlled nuclear Ca 2+ homeostasis. There are InsP 3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP 3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP 3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP 3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP 2, which may have distinct intranuclear functions.

Inositol lipid phosphorylation and breakdown in rat liver nuclei is affected by hydrocortisone blood levels

The possibility that inositol lipid metabolism is related to nuclear events accompanying steroid hormone action has been investigated by comparing lipid phosphorylation and breakdown in normal rat liver nuclei and in hypo- and hypercortisolemic conditions. Lipid phosphorylation in vitro showed the presence of diacylglycerol (DAG)-, phosphatidylinositol (PI)- and phosphatidylinositol-4-phosphate (PIP)-kinase activity, with differences between total tissue homogenates and isolated nuclei, relevant to the treatment in vivo. Administration of hydrocortisone (HC) produced a marked decrease in the phosphorylated nuclear products without influencing the homogenate kinase activity. Under conditions which were optimal for the kinase activities, nuclear PIP-kinase was strongly increased in presence of a high blood level of HC whereas PI-kinase activity was reduced. From these observations it appears that the observed differences were due to specific modulation of kinase activities rather than to changes in the availability of substrates. The phosphoinositide-specific phospholipase C (PLC) activity was also investigated. In the presence of a high HC blood level, the phosphodiesteratic cleavage of PIP strongly increased, while that of phosphatidylinositol bisphosphate (PIP2) was similar in normal and hypercortisolemic conditions. Nuclear phosphoinositide hydrolysis was affected by PLC, beta and gamma isoforms, which were equally represented in all the conditions investigated, indicating that the observed changes of activity were due to a modulation rather than to a change in the amount of enzyme. These results suggest that inositol lipid metabolism plays a role in the nuclear modifications accompanying steroid hormone induction of transcriptional activity.

Interleukin 1 alpha stimulates nuclear phospholipase C in human osteosarcoma SaOS-2 cells.

Interleukin 1 (IL-1) is one of the most potent stimulators of bone resorption. However, the early biochemical events elicited by IL-1 receptor binding are not fully understood. Here we show that in human osteosarcoma SaOS-2 cells the treatment with IL-1 alpha is able to evoke a rapid and transient increase of nuclear phospholipase C (PLC) activity. A parallel decrease of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate is observed. All these events are strictly confined to the nuclear compartment without affecting the cytoplasmatic inositol lipid pool. In addition we show that by Western blot analysis with specific monoclonal antibodies the PLC gamma is located both in the cytoplasm and in the nucleus, while PLC beta appears exclusively localized in the nucleus. Moreover, the increase of PLC activity in response to IL-1 alpha is completely neutralized by monoclonal antibody against the beta-form. While confirming the existence of an autonomous nuclear phosphoinositide signaling system, our data clearly indicate that in SaOS-2 cells one of the earliest events following IL-1 alpha treatment is the breakdown of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate because of the activation of a specific nuclear PLC isoform.

Inositol lipid-mediated intranuclear signalling: A comparative analysis of in vivo labelling in interferon alpha-sensitive and -resistant daudi lymphoma cells

Changes in inositol lipid and diacylglycerol metabolism have been analysed in Daudi lymphoma cells treated up to 24 h with human Dna recombinant interferon alpha. Results showing a different response of nuclear phosphoinositides and diacylglycerol, compared to whole cells, suggest that the intranuclear signalling system activated by interferon in Daudi cells involves nuclear inositol lipid metabolism. A well-characterized clone of Daudi cells selected for resistence to the antiproliferative action of interferon provided controls for the specificity of results.

Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.