Irene Faenza

Phosphatidylinositol 3‐kinase is recruited to a specific site in the activated IL‐1 receptor I

Interleukin 1 (IL‐1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF‐κB. The IL‐1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C‐terminal portion of the type I IL‐1 receptor (IL‐1RI) is essential for IL‐1 signalling and for IL‐1 dependent activation of NF‐κB. This portion contains a putative phosphatidylinositol 3‐kinase (PI 3‐kinase) binding domain (Tyr‐E‐X‐Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3‐kinase in IL‐1 signalling. Here we report evidence that PI 3‐kinase is recruited by the activated IL‐1RI, causing rapid and transient activation of PI 3‐kinase. We also show that the receptor is tyrosine phosphorylated in response to IL‐1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3‐kinase activation by IL‐1. Our results indicate that PI 3‐kinase is likely to be an important mediator of some IL‐1 effects, providing docking sites for additional signalling molecules.

Metabolism and signaling activities of nuclear lipids.

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 10years, 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.

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.

Nuclear pores in the apoptotic cell

SummaryDuring apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.

Involvement of nuclear PLCβ1 in lamin B1 phosphorylation and G2/M cell cycle progression

Inositide‐specific phospholipase Cβ1 (PLCβ1) signaling in cell proliferation has been investigated thoroughly in the G1 cell cycle phase. However, little is known about its involvement in G2/M progression. We used murine erythroleukemia cells to investigate the role of PLCβ1 in G2/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen‐activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G2/M progression. We report that PLCβ1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si‐RNA mediated silencing showed a role of JNK, PKCα, PKCβI, and the β1 isoform of PI‐PLC in cell accumulation in G2/M [as observed by fluorescence‐activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCα,orPLCβ1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCβ1, and, foremost, the finding that the PLCβ1‐mediated pathway represents a functional nuclear inositide signaling in the G2/M transition.— Fiume, R., Ramazzotti, G., Teti, G., Chiarini, F., Faenza, I., Mazzotti, G., Billi, A. M., Cocco, L., Involvement of nuclear PLCβl in lamin B1 phosphorylation and G2/M cell cycle progression. FASEB J. 23, 957–966 (2009)

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.

Molecular characterization of protein kinase C-α binding to lamin A

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)‐binding protein. Here, we have identified the regions of PKC‐α that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione‐S‐transferase (GST) fused to elements of rat PKC‐α, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium‐dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200–217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC‐α to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca2+. We have also found that the binding site of lamin A for the CaLB domain of PKC‐α is localized in the carboxyl‐terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme‐specific drugs to attenuate or reverse PKC‐dependent nuclear signaling pathways important for the pathogenesis of cancer.

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.

Expression of phospholipase C beta family isoenzymes in C2C12 myoblasts during terminal differentiation

In the present work, we have analyzed the expression and subcellular localization of all the members of inositide‐specific phospholipase C (PLCβ) family in muscle differentiation, given that nuclear PLCβ1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLCβ1, β2, β3, β4 have been assessed. As further characterization, we investigated the localization of PLCβ isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCβ4 was the most expressed isoform in the cytoplasm, whereas PLCβ1 and β3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCβ1 isoform was expressed at the highest extent. A marked decrease of PLCβ4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCβ3 at both cytoplasmatic and nuclear level, whilst PLCβ2 expression was almost undetactable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCβ1 and the decrease of cytoplasmatic PLCβ4, during the establishment of myotube differentiation, is related to a spatial‐temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLCβs.

Catalytic activity of nuclear PLC-β1 is required for its signalling function during C2C12 differentiation ☆

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.