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In the last three decades, the presence of phospholipids in the nucleus has been shown and thoroughly investigated. A considerable amount of interest has been raised about nuclear inositol lipids, mainly because of their role in signaling acting. Here, we review the main issues of nuclear phospholipid localization and the role of nuclear inositol lipids and their related enzymes in cellular signaling, both in physiological and pathological conditions.

Polyphosphoinositides (PPIns) are signalling messengers representing less than five per cent of the total phospholipid concentration within the cell. Despite their low concentration, these lipids are critical regulators of various cellular processes, including cell cycle, differentiation, gene transcription, apoptosis and motility. PPIns are generated by the phosphorylation of the inositol head group of phosphatidylinositol (PtdIns). Different pools of PPIns are found at distinct subcellular compartments, which are regulated by an array of kinases, phosphatases and phospholipases. Six of the seven PPIns species have been found in the nucleus, including the nuclear envelope, the nucleoplasm and the nucleolus. The identification and characterisation of PPIns interactor and effector proteins in the nucleus have led to increasing interest in the role of PPIns in nuclear signalling. However, the regulation and functions of PPIns in the nucleus are complex and are still being elucidated. This review summarises our current understanding of the localisation, biogenesis and physiological functions of the different PPIns species in the nucleus.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by peripheral blood cytopenia and abnormal myeloproliferation, as well as a variable risk of evolution into acute myeloid leukemia (AML). The nucleus is a highly organized organelle with several distinct domains where nuclear inositides localize to mediate essential cellular events. Nuclear inositides play a critical role in the modulation of erythropoiesis or myelopoiesis. Here, we briefly review the nuclear structure, the localization of inositides and their metabolic enzymes in subnuclear compartments, and the molecular aspects of nuclear inositides in MDS.

Nuclear inositides have a specific subcellular distribution that is linked to specific functions; thus their regulation is fundamental both in health and disease. Emerging evidence shows that alterations in multiple inositide signalling pathways are involved in pathophysiology, not only in cancer but also in other diseases. Here, we give an overview of the main features of inositides in the cell, and we discuss their potential as new molecular therapeutic targets.

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCβ1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCβ1 and PI-PLCɣ1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCβ1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCβ1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions., (Copyright © 2019 Ratti et al.)

PI-PLCβ1 is involved in cell proliferation, differentiation, and myelodysplastic syndrome (MDS) pathogenesis. Moreover, the increased activity of PI-PLCβ1 reduces the expression of PKC-α, which, in turn, delays the cell proliferation and is linked to erythropoiesis. Lenalidomide is currently used in low-risk patients with MDS and del(5q), where it can suppress the del(5q) clone and restore normal erythropoiesis. In this study, we analyzed the effect of lenalidomide on 16 patients with low-risk del(5q) MDS, as well as del(5q) and non-del(5q) hematopoietic cell lines, mainly focusing on erythropoiesis, cell cycle, and PI-PLCβ1/PKC-α signaling. Overall, 11 patients were evaluated clinically, and 10 (90%) had favorable responses; the remaining case had a stable disease. At a molecular level, both responder patients and del(5q) cells showed a specific induction of erythropoiesis, with a reduced γ/β-globin ratio, an increase in glycophorin A, and a nuclear translocation of PKC-α. Moreover, lenalidomide could induce a selective G 0 /G 1 arrest of the cell cycle in del(5q) cells, slowing down the rate proliferation in those cells. Altogether, our results could not only better explain the role of PI-PLCβ1/PKC-α signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies.-Poli, A., Ratti, S., Finelli, C., Mongiorgi, S., Clissa, C., Lonetti, A., Cappellini, A., Catozzi, A., Barraco, M., Suh, P.-G., Manzoli, L., McCubrey, J. A., Cocco, L., Follo, M. Y. Nuclear translocation of PKC-α is associated with cell cycle arrest and erythroid differentiation in myelodysplastic syndromes (MDSs).

Phosphatidylinositols (PIs) are responsible for several signaling pathways related to many cellular functions, such as cell cycle regulation at different check-points, cell proliferation, cell differentiation, membrane trafficking and gene expression. PI metabolism is not only present at the cytoplasmic level, but also at the nuclear one, where different signaling pathways affect essential nuclear mechanisms in eukaryotic cells. In this review we focus on nuclear inositide signaling in relation to cell cycle regulation. Many evidences underline the pivotal role of nuclear inositide signaling in cell cycle regulation and cell proliferation associated to different strategic physiopathological mechanisms in several cell systems and diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases. In this prospect we analyze the role of PI-PLCβ1 nuclear isoform in relation to the cell cycle regulation, the cell differentiation, and different physiopathological pathways focusing on the importance of the nuclear localization from both molecular and clinical point of view. PI-PLCβ1 is essential for G1/S transition through DAG and Cyclin D3 and plays also a central role in G2/M progression through Cyclin B1 and PKCα. In the differentiation process of C2C12 cells PI-PLCβ1 increases in both myogenic differentiation and osteogenic differentiation. PI-PLCβ1 and Cyclin D3 reduction has been observed in Myotonic Dystrophy (DM) suggesting a pivotal role of these enzymes in DM physiopathology. PI-PLCβ1 is also involved in adipogenesis through a double phase mechanism. Moreover, PI-PLCβ1 plays a key role in the normal hematopoietic differentiation where it seems to decrease in erythroid differentiation and increase in myeloid differentiation. In Myelodysplastic Syndromes (MDS) PI-PLCβ1 has a genetic and epigenetic relevance and it is related to MDS patients' risk of Acute Myeloid Leukemia (AML) evolution. In MDS patients PI-PLCβ1 seems to be also a therapeutic predictive outcome marker. In the central nervous system, PI-PLCβ1 seems to be involved in different pathways in both brain cortex development and synaptic plasticity related to different diseases. Another PI-PLC isozyme that could be related to nuclear activities is PI-PLCζ that is involved in infertility processes. J. Cell. Biochem. 118: 1969-1978, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Lipid signalling molecules are essential components of the processes that allow one extracellular signal to be transferred inside the nucleus, where specific lipid second messengers elicit reactions capable of regulating gene transcription, DNA replication or repair and DNA cleavage, eventually resulting in cell growth, differentiation, apoptosis or many other cell functions. Nuclear inositides are independently regulated, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. Indeed, nuclear inositol lipids themselves can modulate nuclear processes, such as transcription and pre-mRNA splicing, growth, proliferation, cell cycle regulation and differentiation. Nuclear PI-PLCβ1 is a key molecule for nuclear inositide signalling, where it plays a role in cell cycle progression, proliferation and differentiation. Here we review the targets and possible involvement of nuclear PI-PLCβ1 in human physiology and pathology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Inositide signaling pathways can have a role in the Myelodysplastic Syndromes (MDS) progression to acute myeloid leukemia. Erythropoietin (EPO) is currently used in low-risk MDS, where it successfully corrects anemia in 50-70% of patients. However, some MDS patients are refractory to this treatment and little is known about the exact molecular mechanisms underlying the effect of EPO in these subjects. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly focusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, which is activated by the EPO receptor, and PI-PLCbeta1/Cyclin D3 signaling, as Cyclin D3 is associated with hematopoietic proliferation and differentiation. Interestingly, EPO responder patients showed a specific activation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an increase in PI-PLCbeta1 signaling. Moreover, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment.

Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. A number of MDS progresses to acute myeloid leukemia (AML) with the involvement of genetic and epigenetic mechanisms affecting PI-PLC β1. The molecular mechanisms underlying the MDS evolution to AML are still unclear, even though it is now clear that the nuclear signaling elicited by PI-PLC β1, Cyclin D3, and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Moreover, a correlation between other PI-PLCs, such as PI-PLC β3, kinases and phosphatases has been postulated in MDS pathogenesis. Here, we review the findings hinting at the role of nuclear lipid signaling pathways in MDS, which could become promising therapeutic targets.

Myelodysplastic syndromes (MDS) are defined as clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. Although distinct morphologic subgroups exist, the natural history of MDS is progression to acute myeloid leukemia (AML). However, the molecular the mechanisms the underlying MDS evolution to AML are not completely understood. Inositides are key cellular second messengers with well-established roles in signal transduction pathways, and nuclear metabolism elicited by phosphoinositide-specific phospholipase C (PI-PLC) beta1 and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Recent findings evidenced the role played by nuclear lipid signaling pathways, which could become promising therapeutic targets in MDS. This review will provide a concise and updated revision of the state of art on this topic.

Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

Phosphoinositides (PI) are the most extensively studied lipids involved in cell signaling pathways. The bulk of PI is found in membranes where they are substrates for enzymes, such as kinases, phosphatases and phospholipases, which respond to the activation by cell-surface receptors. The outcome of the majority of signaling pathways involving lipid second messengers results in nuclear responses finally driving the cell into differentiation, proliferation or apoptosis. Some of these pathways are well established, such as that of PI-specific phospholipase C (PI-PLC), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) into the two second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). Two independent cycles of PI are present inside the cell. One is localized at the plasma membrane, while the most recently discovered PI cycle is found inside the nuclear compartment. The regulation of the nuclear PI pool is totally independent from the plasma membrane counterpart, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. In this report we will focus on the signal transduction-related metabolism of nuclear PI and review the most convincing evidence that the PI cycle is involved in differentiation programs in several cell systems.

The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG-dependent events. It has been demonstrated that nuclear DGK-zeta interferes with cell cycle progression. We previously reported that nuclear DGK-zeta expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK-zeta in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-zeta, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-16-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK-zeta by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK-zeta was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-zeta, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK-zeta as a key determinant of cell cycle progression and differentiation of C2C12 cells.

Lipid second messengers, particularly those derived from the polyphosphoinositide metabolism, play a pivotal role in multiple cell signaling networks. Phosphoinositide 3-kinase (PI3K) generate 3'-phosphorylated inositol lipids that are key players in a multitude of cell functions. One of the best characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt (protein kinase B, PKB). Recent findings have implicated the PI3K/Akt pathway in tumorigenesis because it stimulates cell proliferation and suppresses apoptosis. However, it was thought that this signal transduction network would exert its carcinogenetic effects mainly by operating in the cytoplasm. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid cycle, and strongly suggests that lipid molecules are important components of signaling pathways operating at the nuclear level. PI3K, its lipid product phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3), and Akt have been identified within the nucleus and recent data suggest that they counteract apoptosis also by operating in this cell compartment through a block of caspase-activated DNase and inhibition of chromatin condensation. In this review, we shall summarize the most updated and intriguing findings about nuclear PI3K/PtdIns(3,4,5)P3/Akt in relationship with tumorigenesis and suppression of apoptotic stimuli.

A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC beta1 is a well-known example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC beta1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, non-specific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC beta1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC beta1 gene. In contrast, PLC beta4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC beta1, is unaffected in MDS patients with the deletion of PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC beta1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC beta1 monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC beta1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC beta1. The reported data strengthen the contention of a key role played by PLC beta1 in the nucleus, suggest a possible involvement of PLC beta1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.

Previous evidence from independent laboratories has shown that the nucleus contains diacylglycerol kinase (DGK) isoforms, i.e., the enzymes, which yield phosphatidic acid from diacylglycerol, thus terminating protein kinase C-mediated signaling events. A DGK isoform, which resides in the nucleus of PC12 cells, is DGK-theta. Here, we show that nerve growth factor (NGF) treatment of serum-starved PC12 cells results in the stimulation of both a cytoplasmic and a nuclear DGK activity. However, time course analysis shows that cytoplasmic DGK activity peaked earlier than its nuclear counterpart. While nuclear DGK activity was dramatically down-regulated by a monoclonal antibody known for selectively inhibiting DGK-theta, cytoplasmic DGK activity was not. Moreover, nuclear DGK activity was stimulated by phosphatidylserine, an anionic phospholipid that had no effect on cytoplasmic DGK activity. Upon NGF stimulation, the amount and the activity of DGK-theta, which was bound to the insoluble nuclear matrix fraction, substantially increased. Epidermal growth factor up-regulated a nuclear DGK activity insensitive to anti-DGK-theta monoclonal antibody. Overall, our findings identify nuclear DGK-theta as a down-stream target of NGF signaling in PC12 cells.

Apart from the lipids present in the nuclear envelope, the nucleus also contains lipids which are located further inside and are resistant to treatment with nonionic detergents. Evidence is being accumulated on the importance of internal nuclear lipid metabolism. Nuclear lipid metabolism gives rise to several lipid second messengers that function within the nucleus. Moreover, it is beginning to emerge that nuclear lipids not only act as precursors of bioactive second messengers but may be directly involved in regulation of nuclear structure and gene expression. Over the last 10 years, especially the role of the inositol lipid cycle in nuclear signal transduction has been extensively studied. This cycle is activated following a variety of stimuli and is regulated independently from the inositide cycle located at the plasma membrane. However, the nucleus contain other lipids, such as phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. There are numerous reports which suggest that these classes of nuclear lipids may play roles in the nucleus as important as those of phosphoinositides. This review aims at highlighting the most important aspects regarding the metabolism and signaling activities of nuclear phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids.

Strong evidence has been accumulating over the last 15 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. Several advances have resulted in the discovery that nuclear phosphoinositides are involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Although nuclear inositol lipids generate second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and updated findings about inositol lipid metabolism in the nucleus.

A body of evidence, linking inositide-specific phospholipase C (PI-PLC) to the nucleus, is quite extensive. The main isoform in the nucleus is PI-PLCbeta1, whose activity is up-regulated in response to insulin-like growth factor-1 (IGF-1) or insulin stimulation. Whilst at the plasma membrane this PI-PLC is activated and regulated by Galphaq/alpha(11) and Gbetagamma subunits, there is yet no evidence that qalpha/alpha(11) is present within the nuclear compartment, neither GTP-gamma-S nor AlF4 can stimulate PI-PLCbeta1 activity in isolated nuclei. Here we review the evidence that upon occupancy of type 1 IGF receptor there is translocation to the nucleus of phosphorylated mitogen-activated protein kinase (MAPK) which phosphorylates nuclear PI-PLCbeta1 and triggers its signalling, hinting at a separate pathway of regulation depending on the subcellular location of PI-PLCbeta1. The difference in the regulation of the activity of PI-PLCbeta1mirrors the evidence that nuclear and cytoplasmatic inositides can differ markedly in their signalling capability. Indeed, we do know that agonists which affect nuclear inositol lipid cycle at the nucleus do not stimulate the one at the plasma membrane.

Protein kinase C (PKC) isozymes are a family of serine/threonine protein kinases categorized into three subfamilies: classical, novel, and atypical. PKC isozymes, whose expression is cell type-specific and developmentally regulated, are key transducers in many agonist-induced signaling cascades. To date at least 10 different PKC isotypes have been identified and are believed to play distinct regulatory roles. PKC isoforms are catalytically activated by several lipid cofactors, including diacylglycerol. PKC is thought to reside in the cytoplasm in an inactive conformation and to translocate to the plasma membrane or cytoplasmic organelles upon cell activation by different stimuli. However, a sizable body of evidence collected over the last 15 years has shown PKC to be capable of translocating to the nucleus. Furthermore, PKC isoforms can reside within the nucleus. Studies from independent laboratories have to led to the identification of several nuclear proteins which act as PKC substrates as well as to the characterization of some nuclear PKC-binding proteins which may be of fundamental importance for finely tuning PKC function in this peculiar cell microenvironment. Most likely, nuclear PKC isozymes are involved in the regulation of several important biological processes such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we shall summarize the most intriguing evidence about the roles played by nuclear PKC isozymes.

Growth, differentiation, and apoptosis of eukaryotic cells are mediated by extremely complex signaling pathways and a high degree of coordination is required for regulating cell proliferation. In multicellular organisms homeostasis is achieved through signal transduction events. If these homeostatic mechanisms are interrupted, a disease, such as cancer, may ensue. Lipid second messengers, particularly those derived from polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signaling molecules are important components of signaling pathways operating within the nucleus. Recent findings are starting to elucidate how the nuclear phosphoinositide cycle is regulated and what down-stream molecules are targeted through this cycle. In this review, we shall summarize the most updated data about inositol lipid-dependent nuclear signaling pathways that might have a relevance for the development of cancer. In the near future, this knowledge might also prove to have relevance for the diagnosis and treatment of the neoplastic disease.

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.

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.

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.

The role of polyphosphoinositides in cellular signalling is well known and recently it has also been shown that the nucleus is a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. It has been demonstrated that phospholipase C specific for inositol lipids (PLC) is one of the main steps of the inositol lipid cycle. The PLC beta family, and especially type beta 1, has given rise to considerable interest since, due to their common COOH-terminus they show nuclear localisation in addition to that at the plasma membrane. It is well established that an autonomous intranuclear inositide cycle exists, and that this cycle is endowed with conventional lipid kinases, phosphatases and PLCs. Among this latter the beta 1 type undergoes stimulation or inhibition under different stimuli and this implicates the beta 1 isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. Indeed, both the overexpression and the down-regulation of PLC beta 1, by means of antisense mRNA, have demonstrated that PLC plays a role in the nuclear compartment.

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.

In order to evaluate at the ultrastructural level the chromatin arrangement during the S phase of the cell cycle, the detection of Bromodeoxyuridine (BrdU) by immunogold has been performed in synchronized 3T3 fibroblasts, regenerating liver, and Friend Leukemia Cells (FLC). After a 5-minute BrdU pulse, this label is detected in 10-nm-wide fibers, organized as lacework and assumed to be replication units. In the early part of the S phase, DNA replication units are localized exclusively in the dispersed chromatin domains far from the nuclear envelope. In the middle S, replication occurs at the border between condensed and dispersed chromatin and, finally, in late S, it mainly occurs in perinuclear heterochromatin regions. After replication, the 10-nm fibers can condense in heterochromatin without translocation. Chromatin is highly dispersed in early S and computer image analysis shows an increase in condensed chromatin areas ranging from 13 to 18% at the end of the S phase with a temporal and morphological pattern of distribution characteristic for each cell type. Scanning transmission electron microscopy demonstrates a regular and repetitive structure of dispersed chromatin, represented by a ring-like arrangement of the 10-nm fibers; assuming the same spatial distribution, gold particles that identify incorporated BrdU confirm this organization. By evaluating the organization and the distribution of DNA replication units during S phase, the results suggest that DNA replication occurs at a nucleosomal-like fiber level and that replicating enzymes machinery moves over a fixed template.

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 beta1 (PLC beta1) 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 beta1., (Copyright 1997 Academic Press.)

The nucleus has been shown to be a site for the inositol lipid cycle that can be affected by treatment of quiescent cells with growth factors such as insulin-like growth factor I (IGF-I). Indeed, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear phospholipase C (PLC) beta1 activity. In addition, several other reports have shown the involvement of PLC beta1 in nuclear signaling in different cell types. Although the demonstration of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis by nuclear PLC beta1 established the existence of nuclear PLC signaling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta1 expression by antisense RNA and its overexpression, we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells.

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-TP alpha isoform. Upon induction of erythroid differentiation by DMSO, the amount of nuclear PI-TP alpha was greatly diminished. As shown previously, under these same conditions, nuclear phospholipase C beta1 (PLC beta1) is down-regulated as well.

The involvement of phospholipids and especially polyphosphoinositides in cellular signalling has been documented in detail over the last 20 years. Besides the membrane localisation the nucleus also has been show to be a site for both the synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observations dealing with signal transduction have established phospholipase C, specific for inositol lipids (PLC), an important step in the inositol lipid cycle. Of the several known PLC isoforms the type beta 1 is of particular interest because of its reported nuclear localisation, in addition to its presence at the plasma membrane. Indeed, investigations from our laboratory and others have shown the existence in several cell types of an autonomous intranuclear inositide cycle endowed with both conventional lipid kinases and PLC. Moreover, both the stimulation and the inhibition of the nuclear PLC beta 1 under different stimuli implicate this PLC isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. These findings have prompted us to better characterise the nuclear PLC beta 1. The isoform beta 1 has been studied as a possible target for anti-cancer drugs and as an inducer, via diacylglycerol generation, of the translocation of specific protein kinase C (PKC) isozyme to the nucleus. The chromosome mapping of PLC beta 1 gene has been carried out and the effect of its knock-out by means of antisense cDNA has been determined.

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.

Previous investigations have demonstrated the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and phospholipase C (PLC) which is the isoform beta in Swiss 3T3 cells, PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, and rat liver. The presence of PLC has been investigated in nuclei of Friend erythroleukemia cells. Both beta and gamma isoforms are present in these nuclei. When Friend cells undergo terminal erythroid differentiation in the presence of dimethyl sulfoxide the PLC beta isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by determination of enzymatic activity directly and in the presence of neutralizing monoclonal antibodies and also by Northern blot for PLC beta message. By contrast, the amount of PLC gamma and its activity are unaffected by erythroid differentiation. Thus, the presence of a nuclear PLC beta, the activity and expression of which are modulated during differentiation of erythroleukemia cells, implicates a role for nuclear phosphoinositide signaling in the processes of cell determination and indicates the nuclear PLC beta as a key enzyme of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.

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.

Two different clones of Swiss 3T3 cells belonging to the same original cell line have been obtained, one of which was unresponsive to mitogenic stimulation (e.g. insulin-like growth factor-I, bombesin, insulin-like growth factor-I + bombesin), while the other clone showed a very high rate of DNA synthesis under identical conditions as demonstrated by 5-bromodeoxyuridine incorporation. Both types of cells expressed the IGF-I receptor and showed high contact inhibition. When highly purified nuclei from responsive cells, treated for a short time with bombesin and insulin-like growth factor-I or insulin-like growth factor-I alone, were incubated with [gamma-32P]adenosine triphosphate, the labelling of phosphatidylinositol-mono- and diphosphate decreased when compared to controls, while this transient effect did not appear in the nuclei from unresponsive cells. Similarly nuclear protein kinase C is activated only in responsive cells. Therefore, it seems that a direct link exists between polyphosphoinositide metabolism, protein kinase C activation and the early events leading to cell division, since the rapid changes in the labelling of both phosphatidylinositol mono- and di-phosphate occur only in nuclei from Swiss 3T3 cells, which respond to the mitogenic stimulus determined by insulin-like growth factor-I on its own, or in combination with bombesin.

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.

Since phospholipids have been suggested to play some role in the molecular organisation of the nuclear matrix, the effect of their removal by means of phospholipase C has been investigated in regenerating rat liver nuclear matrix. The matrix-bound DNA polymerase alpha shows an almost complete loss of activity following the digestion with phospholipase C. Since the polymerase activity is restored by adding exogenous DNA, we suggest that the effect is due to the removal of matrix residual DNA, which is in some way linked to the nuclear matrix by means of hydrophobic interactions.

The water-soluble probe carboxyfluorescein (CF), contained in the internal aqueous phase of liposomes, was used to investigate the interaction of phospholipid vesicles with isolated nuclei. Ultrastructural analysis indicated that adherent liposomes coated the nuclear surface, and fluorescence microscopy showed that they contained quenching concentrations of the dye. Flow cytometry revealed that the transfer of the entrapped dye from the adhering liposomes to nuclei was blocked by chilling at 0 degrees C. Chase experiments demonstrated that the most reliable mechanism of dye transfer involved fusion phenomena between the liposomal and the nuclear membranes. After the release of the fluorophore into the nucleus, empty liposomes could withdraw the intranuclear soluble fraction of the dye.