Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

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.

Essential role for nuclear phospholipase C beta1 in insulin-like growth factor I-induced mitogenesis.

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.

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-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.

Control of expression of PLCbeta1 by LAC repressor system: relationship between nuclear PLCbeta1 overexpression 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 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.

Phosphoinositide signaling in nuclei of Friend cells: tiazofurin down-regulates phospholipase C beta 1.

Previous investigations have demonstrated the presence of conventional lipid kinases and phospholipase C (PLC) activities in nuclei of Friend erythroleukemia cells. Moreover, when Friend erythroleukemia cells are treated for 96 h with the antitumor drug tiazofurin, the induction of erythroid differentiation is accompanied by changes in amounts of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate due to the inhibition of an uncharacterized nuclear PLC activity. Here, we show that the nuclear PLC beta 1 isoform is down-regulated by tiazofurin (5 microM) treatment of Friend erythroleukemia cells as shown by both Western blot and Northern blot analyses for PLC beta 1 message. This indicates that PLC beta 1 down-regulation is tightly linked with erythroid differentiation of Friend erythroleukemia cells and that the autonomous nuclear signaling via inositol lipid cycle can be controlled by the antitumor drug tiazofurin.

Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation.

Friend erythroleukemia cells were labelled with high levels of [3H]myo-inositol and the radioactivity in PI, PIP and PIP2, extracted from isolated nuclei, was measured. A parallel analysis employing a picomole sensitive assay for both PIP and DAG has been carried out. The results indicate that the differentiation process is characterised by an accumulation of nuclear PIP and PIP2 and by a decrease of DAG mass. We suggest that as differentiation proceeds toward erythrocytes in Friend cells, this is accompanied by a reduction in the amount of these messengers in the nucleus.

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins.

Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at 0 degrees C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37 degrees C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37 degrees C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37 degrees C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37 degrees C in vitro, unlike to that what previous reports have indicated.

Ultrastructural organization of the sperm nuclear matrix.

The organization of DNA-protamine complexes and their association with the nuclear matrix have been analyzed in sperm nuclei by in situ Nick Translation at the electron microscope. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interaction with the nuclear matrix at fixed sites. The fine structure of the sperm nucleus and sperm nuclear matrix, investigated by sectioning and replica of freeze-fractured specimens, suggests that the lamellar array observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures involved in the maintenance of chromatin domains.

Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF.

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.

Nuclear matrix involvement in sperm head structural organization.

In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNA-protamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine structure of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.