G. Ramazzotti | AICCON - Associazione Italiana per la Promozione della Cultura della Cooperazione e del Non Profit (original) (raw)

Papers by G. Ramazzotti

Molecular & Cellular Proteomics, Oct 5, 2010

Recent data indicate that some PKC isoforms are translocated to the nucleus, in response to certa... more Recent data indicate that some PKC isoforms are translocated to the nucleus, in response to certain stimuli, where they play an important role in nuclear signaling events. To identify novel interacting proteins of conventional PKC (cPKC) at the nuclear level during myogenesis and to find new PKC isozyme-specific phosphosubstrates, we performed a proteomics analysis of immunoprecipitated nuclear samples from mouse myoblast C2C12 cells following insulin administration. Using a phospho(Ser)-PKC substrate antibody, specific interacting proteins were identified by LC-MS/MS spectrometry. A total of 16 proteins with the exact and complete motif recognized by the phospho-cPKC substrate antibody were identified; among these, particular interest was given to eukaryotic elongation factor 1␣ (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the cPKC isoforms, only PKC␤I was demonstrated to be expressed in the nucleus of C2C12 myocytes and to coimmunoprecipitate with eEF1A. In-depth analysis using site-directed mutagenesis revealed that PKC␤I could phosphorylate Ser 53 of the eEF1A2 isoform and that the association between eEF1A2 and PKC␤I was dependent on the phosphorylation status of eEF1A2.

The FASEB Journal, 2009

Inositide-specific phospholipase Cbeta1 (PLCbeta1) signaling in cell proliferation has been inves... more Inositide-specific phospholipase Cbeta1 (PLCbeta1) signaling in cell proliferation has been investigated thoroughly in the G(1) cell cycle phase. However, little is known about its involvement in G(2)/M progression. We used murine erythroleukemia cells to investigate the role of PLCbeta1 in G(2)/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 G(2)/M progression. We report that PLCbeta1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si-RNA mediated silencing showed a role of JNK, PKCalpha, PKCbetaI, and the beta1 isoform of PI-PLC in cell accumulation in G(2)/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, PKCalpha, or PLCbeta1 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 PLCbeta1, and, foremost, the finding that the PLCbeta1-mediated pathway represents a functional nuclear inositide signaling in the G(2)/M transition.

The FASEB Journal, 2012

Type 2 diabetes is a heterogeneous disorder caused by concomitant impairment of insulin secretion... more Type 2 diabetes is a heterogeneous disorder caused by concomitant impairment of insulin secretion by pancreatic ␤ cells and of insulin action in peripheral target tissues. Studies with inhibitors and agonists established a role for PLC in the regulation of insulin secretion but did not distinguish between effects due to nuclear or cytoplasmic PLC signaling pathways that act in a distinct fashion. We report that in MIN6 ␤ cells, PLC␤1 localized in both nucleus and cytoplasm, PLC␦4 in the nucleus, and PLC␥1i nt h e cytoplasm. By silencing each isoform, we observed that they all affected glucose-induced insulin release both at basal and high glucose concentrations. To elucidate the molecular basis of PLC regulation, we focused on peroxisome proliferator-activated receptor-␥ (PPAR␥), a nuclear receptor transcription factor that regulates genes critical to ␤-cell maintenance and functions. Silencing of PLC␤1 and PLC␦4 resulted in a decrease in the PPAR␥ mRNA level. By means of a PPAR␥promoter-luciferase assay, the decrease could be attributed to a PLC action on the PPAR␥-promoter region. The effect was specifically observed on silencing of the nuclear and not the cytoplasmic PLC. These findings highlight a novel pathway by which nuclear PLCs affect insulin secretion and identify PPAR␥ as a novel molecular target of nuclear PLCs.-Fiume, R., Ramazzotti, G., Faenza, I., Piazzi, M., Bavelloni, A., Billi, A. M., Cocco, L. Nuclear PLCs affect insulin secretion by targeting PPAR␥ in pancreatic ␤ cells. FASEB J. 26, 000 -000 (2012). www.fasebj.org

Proceedings of the National Academy of Sciences, 2009

Lipid signaling pathways are involved in cell growth, differentiation, and apoptosis, and could h... more Lipid signaling pathways are involved in cell growth, differentiation, and apoptosis, and could have a role in the progression of myelodysplastic syndromes (MDS) into acute myeloid leukemia (AML). Indeed, recent studies showed that phosphoinositide-phospholipase (PI-PL)Cbeta1 mono-allelic deletion correlates with a higher risk of AML evolution. Also, a single patient treated with azacitidine, a DNA methyltransferase inhibitor currently used in MDS, displayed a direct correlation between PI-PLCbeta1 gene expression and drug responsiveness. Consequently, we hypothesized that PI-PLCbeta1 could be a target for demethylating therapy. First, we analyzed the structure of PI-PLCbeta1 gene promoter, then quantified the degree of PI-PLCbeta1 promoter methylation and gene expression in MDS patients at baseline and during azacitidine administration. Indeed, PI-PLCbeta1 mRNA increased in responder patients, along with a reduction of PI-PLCbeta1 promoter methylation. Also, the molecular response correlated to and anticipated the clinical outcome, thus suggesting that PI-PLCbeta1 gene reactivation could predict azacitidine responsiveness. Our results demonstrate not only that PI-PLCbeta1 promoter is hypermethylated in high-risk MDS patients, but also that the amount of PI-PLCbeta1 mRNA could predict the clinical response to azacitidine, therefore indicating a promising new therapeutic approach.

Leukemia, 2011

The association between azacitidine (AZA) and valproic acid (VPA) has shown high response rates i... more The association between azacitidine (AZA) and valproic acid (VPA) has shown high response rates in high-risk myelodysplastic syndromes (MDS) cases with unfavorable prognosis. However, little is known about the molecular mechanisms underlying this therapy, and molecular markers useful to monitor the disease and the effect of the treatment are needed. Phosphoinositide-phospholipase C (PI-PLC) b1 is involved in both genetic and epigenetic mechanisms of MDS progression to acute myeloid leukemia. Indeed, AZA as a single agent was able to induce PI-PLCb1 expression, therefore providing a promising new tool in the evaluation of response to demethylating therapies. In this study, we assessed the efficacy of the combination of AZA and VPA on inducing PI-PLCb1 expression in high-risk MDS patients. Furthermore, we observed an increase in Cyclin D3 expression, a downstream target of PI-PLCb1 signaling, therefore suggesting a potential combined activity of AZA and VPA in high-risk MDS in activating PI-PLCb1 signaling, thus affecting cell proliferation and differentiation. Taken together, our findings might open up new lines of investigations aiming at evaluating the role of the activation of PI-PLCb1 signaling in the epigenetic therapy, which may also lead to the identification of innovative targets for the epigenetic therapy of high-risk MDS.

Journal of Cellular Physiology, 2011

Nuclear inositide signaling is nowadays a well-established issue and a growing field of investiga... more Nuclear inositide signaling is nowadays a well-established issue and a growing field of investigation, even though the very first evidence came out at the end of the 1980&amp;amp;amp;amp;amp;amp;#x27;s. The understanding of its biological role is supported by the recent acquisitions dealing with pathology and namely hematological malignancies. Here, we review this issue highlighting the main achievements in the last years.

Endocrinology, 2007

Our main goal in this study was to investigate the role of phospholipase C (PLC) ␤ 1 and PLC␥ 1 i... more Our main goal in this study was to investigate the role of phospholipase C (PLC) ␤ 1 and PLC␥ 1 in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLC␤ 1 in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLC␤ 1 or PLC␥ 1 cDNA and with small interfering RNAs from regions of the PLC␤ 1 or PLC␥ 1 gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLC␤ 1 and PLC␥ 1 were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLC␤ 1 or PLC␥ 1 expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLC␤ 1 or PLC␥ 1 cDNAs and small interfering RNAs, respectively. Our data indicate that PLC␤ 1 or PLC␥ 1 signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLC␤ 1 and PLC␥ 1 during myogenic differentiation in vitro and implies that up-regulation of these enzymes is sufficient to mimic the actions of insulin in this process. (

Future Lipidology, 2007

... Role of nuclear PLC and PI3K signaling in the development of cancer. Irene Faenza , Giulia Ra... more ... Role of nuclear PLC and PI3K signaling in the development of cancer. Irene Faenza , Giulia Ramazzotti , Alberto Bavelloni , Lisa Bregoli , Roberta Fiume , Gian Carlo Gaboardi ... Lachyankar MB, Sultana N, Schonhoff CM et al.: A role for nuclear PTEN in neuronal differentiation. ...

Cellular Signalling, 2010

In a previous report we have demonstrated that PLCγ1 is involved in the differentiation process o... more In a previous report we have demonstrated that PLCγ1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCγ1dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCε and η expression, and that PKCε is able to form a complex with PLCγ1. The increase in PKCε amount during myogenic differentiation is associated to an increase in PKCε activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCγ1 and PKCε localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCγ1 and PKCε increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCγ1-PKCε complex co-localizes with protein 58 K, a specific Golgi marker. Moreover, our results indicate that the Golgiassociated PKCε form, i.e. PKCε phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCγ1, we analyzed the effect on cyclin D3 expression of either PKCε overexpression or silencing, in order to investigate whether PKCε could also affect cyclin D3 expression. The results showed that either a modification of PKCε expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCε in regulating insulin inositide-dependent PLCγ1 signalling in skeletal muscle differentiation.

Cellular Signalling, 2008

Here we report that PLC-β 1 catalytic activity plays a role in the increase of cyclin D3 levels a... more Here we report that PLC-β 1 catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-β 1 mutational analysis revealed the importance of His 331 and His 378 for the catalysis. The expression of PLC-β 1 and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-β 1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β 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-β 1 catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-β 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-β 1 and that c-Jun binding activity is significantly increased by insulin and PLC-β 1 overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-β 1 . These results hint at the fact that PLC-β 1 catalytic activity signals a c-jun/ AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

Advances in Enzyme Regulation, 2008

ABSTRACT Here we have reviewed the role of nuclear PLCβ1 signaling and namely its down-stream tar... more ABSTRACT Here we have reviewed the role of nuclear PLCβ1 signaling and namely its down-stream targets as well as the significance of inositide signaling, basically in the nucleus, in MDS blasts. As concerning myogenic differentiation, PLCβ1 along with the γ1 isoform, is essential for C2C12 myoblasts to become myotubes as judged by both molecular and morphological analysis. Both PLCs are capable of inducing, once overexpressed in their physiological compartment, the differentiation of C2C12 myoblasts by targeting specific regions of the cyclin D3 promoter even in the absence of insulin. When dealing with MDS it has been found that nuclear PLCβ1 has a specific role in MDS blasts and this is not only because of the presence of the mono-allelic deletion of its gene in high-risk patients who evolved in AML, but also because of its modulation, namely up-regulation, by means of azacitidine in high-risk responsive patients, which parallels the decrease of activated Akt. Indeed this seems interesting in the light of the fact that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients, and that this signaling network could become an interesting therapeutic target for treating more advanced MDS patients.

Current Medicinal Chemistry, 2007

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamyci... more The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.