R. Stewart Gilmour

Up-regulation of nuclear PLCβ1 in myogenic differentiation

Phospholipase C β1 (PLCβ1) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCβ1, i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCβ1a and PLCβ1b. Indeed, treatment with insulin induces a dramatic rise of both PLCβ1 subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti‐PLCβ1 specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCβ1 expression versus Troponin T expression clearly indicates that the increase in the amount of PLCβ1 takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCβ1M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCβ1 is a key player in myoblast differentiation, functioning as a positive regulator of this process.

Insulin selectively stimulates nuclear phosphoinositide‐specific phospholipase C (PI‐PLC) β1 activity through a mitogen‐activated protein (MAP) kinase‐dependent serine phosphorylation

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 β family of PI‐PLC (i.e. β1, β2, β3, and β4), insulin only activated the β1 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 [32P]orthophosphate, we ascertained that nuclear PI‐PLC‐β1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI‐PLC‐β1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI‐PLC‐β1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.

Nuclear PLCβ1 acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells

It is well established that phospholipase C (PLC) h1 plays a role in the nuclear compartment and is involved in the signalling pathway that controls the switching of the erythroleukemia cells programming from an undifferentiated to a differentiated state. Constitutive overexpression of nuclear PLCh1 has been previously shown to inhibit Friend cells differentiation. For further characterization, we investigated the localization of PLCh1a and PLCh1b in Friend cells by fusing their cDNA to enhanced green fluorescent protein (GFP). To investigate the potential target of nuclear PLCh1 in Friend differentiation, we studied the expression of p45/NF-E2 transcription factor, which is an enhancer binding protein for expression of the h-globin gene and the expression of GATA proteins that are important for the survival and differentiation of erythroid cells. Our data suggest that the overexpression of PLCh1 (both 1a and 1b) only in the nuclear compartment significantly reduces the expression of p45/NF-E2. The effect observed is attributable to the specific action of nuclear PLCh1 signalling given that GATA-1 and GATA-3 are not affected at all. Here we show the existence of a unique target, i.e. the transcription factor p45/NF-E2, whose expression is specifically inhibited by the nuclear signalling evoked by PLCh1 forms. D 2002 Elsevier Science B.V. All rights reserved.

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase β 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.

Nuclear lipid signaling: novel role of eicosanoids.

Nuclear lipid signaling is an established, widespread mechanism that operates in multiple cellular processes including proliferative and differentiative responses to a variety of stimuli. In this literature review with key references highlighted, we put forward the hypothesis that differential flow through various intracrine mechanisms can dictate resultant cellular actions such as mitosis, differentiation, or apoptosis.

Inositides in the nucleus: taking stock of PLCβ1

The nucleus was shown to be a site for inositol lipid cycle which can be affected by treatment of quiescent cells with growth factors such as IGF-I. In fact, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear PLC beta 1 activity. In addition, several other reports have shown the involvement of PLC beta 1 in nuclear signalling in different cell types. Indeed, PLC beta 1 differs from the PLC gamma and della isozymes in that it has a long COOH-terminal sequence which contains a cluster of lysine residues that are critical for association with the nucleus. Although the demonstration of PtInsP and PtdInsP2 hydrolysis by nuclear PLC beta 1 established the existence of nuclear PLC signalling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta 1 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. Moreover, using a different cell system, i.e. Friend erythroleukemia cells induced to differentiate towards erythrocytes, it has been evidenced that there is a relationship between the expression and activity of nuclear PLC beta 1 and the association of PI-PT alpha with the nucleus in that, when PLC activity ceases, in differentiated and resting cells at the same time there is a dramatic decrease of the association of PI-PT alpha with the nucleus.

Inositides in the nucleus: further developments on phospholipase Cβ1 signalling during erythroid differentiation and IGF-I induced mitogenesis

Inositol lipids originally shown to be metabolized in the cytosol have been detected also in the nucleus, where they are both synthesized and hydrolyzed. In the case of erythroid differentiation of murine erythroleukemia cells (Friend cells) it has been previously shown that PLC beta 1, which is the major nuclear PLC, undergoes down-regulation upon treatment with DMSO or tiazofurin which act as differentiative agents. On the contrary, i.e., during IGF-I induced mitogenesis, it has been shown that PLC beta 1 is rapidly activated and this event is essential for the onset of DNA synthesis. Even though its key role in cell growth has been shown, both the mechanism by which nuclear PLC beta 1 is activated and the direct relationship with erythroid differentiation are still unknown. We have addressed the question if PLC beta 1 expression and activity in the nucleus are directly related or not to the establishment of the differentiated state and we have checked the two main ways of activation, i.e., via G-protein or via phosphorylation, in order to establish whether nuclear PLC beta 1 is regulated the same way as the one at the plasma membrane or not. The data reported here show that nuclear PLC beta 1 is responsible for a continuous recycling of Friend cells, acting as a negative regulator of differentiation and that its activation is dependent on the phosphorylation state.