Roberta Fiume - Academia.edu (original) (raw)
Papers by Roberta Fiume
Italian journal of anatomy and embryology, 2017
Italian journal of anatomy and embryology, 2015
Italian journal of anatomy and embryology, 2016
The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characteri... more The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections. The evidence of a clinical correlation between the presence of a monoallelic gene deletion of Phospholipase Cβ1 (PLCβ1) and the progression of MDS to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments. Patients affected by MDS with a higher risk of AML evolution have a reduction in the expression of the nuclear PLCβ1, which is also epigenetically relevant in MDS. This strengthens the importance of PLCβ1 localization. In fact, PLCβ1 is a molecular target for hypomethylating agents, such Azacitidine (AZA)(1). High-risk MDS patients that respond to the drug showed an increased expression of nuclear PLCβ1 and its downstream target Cyclin D3 (CCND3), an induction of normal myeloid differentiation, and a better prognosis. Stemming from these data, ...
Handbook of Experimental Pharmacology, 2019
Nuclear inositides have a specific subcellular distribution that is linked to specific functions;... more 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.
Journal of Lipid Research, 2018
Nuclear phosphoinositide-specific phospholipases C (PI-PLCs) are a group of enzymes that hydrolyz... more Nuclear phosphoinositide-specific phospholipases C (PI-PLCs) are a group of enzymes that hydrolyze phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] to inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) that, in turn, are key second messengers involved in the activation of several signaling pathways related to many critical cellular functions (1, 2). Indeed, nuclear PI-PLCs play pivotal roles in cell cycle regulation (3, 4), cell proliferation, cell differentiation, membrane trafficking, and gene expression (5, 6). Recent evidence on the role of nuclear PI-PLC signaling in cell cycle regulation and cell proliferation paved the way to new fields of research related to different strategic physiopathological mechanisms in many cellular systems and diseases. This is the case of PI-PLC1, which plays a role in the physiopathology of brain disorders (along with PI-PLC1), hematological malignancies, and neuromuscular diseases, but also of PI-PLC-, which has been associated with fertility disorders. All in all, the imbalance of nuclear PI-PLC isoenzymes, such as PI-PLC1 and PI-PLC-, can lead to pathology (7).
Advances in biological regulation, 2017
Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the geneti... more Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation and osteogenesis. During myogenic differentiation of murine C2C12 myoblasts, PLC-β1 signaling pathway involves the Inositol Polyphosphate Multikinase (IPMK) and β-catenin as downstream effectors. By means of c-jun binding to cyclin D3 promoter, the activation of PLC-β1 pathway determines cyclin D3 accumulation. However, osteogenesis requires PLC-β1 expression and up-regulation but it does not affect cyclin D3 levels, suggesting that the two processes require the activation of different mediators.
Italian journal of anatomy and embryology, 2012
Oncotarget, Jan 30, 2014
PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin ... more PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCβ1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCβ1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCβ1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCβ1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCβ1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effect...
Advances in biological regulation, 2013
Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older ... more 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. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia.
Advances in biological regulation, 2012
International Journal of Molecular Medicine, 2006
Phosphoinositide-specific phospholipase C (PI-PLC) ß1 is a key enzyme in nuclear signal transduct... more Phosphoinositide-specific phospholipase C (PI-PLC) ß1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLCß1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLCß1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLCß1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCß1. To analyze and quantify the levels of the two different splicing variants of PI-PLCß1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PI-PLCß1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLCß1b mRNA compared to PI-PLCß1a mRNA. Our data support the contention that the deletion of the PI-PLCß1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the 1a isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.
FEBS Journal, 2013
Evidence accumulated over the past 20 years has highlighted the presence of an autonomous nuclear... more Evidence accumulated over the past 20 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signalling molecules are important components of signalling pathways operating within the nucleus. Nuclear polyphosphoinositide (PI) signalling relies on the synthesis and metabolism of phosphatidylinositol 4,5-bisphosphate, which can modulate the activity of effector proteins and is a substrate of signalling enzymes. 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. Among the nuclear enzymes involved in PI metabolism, inositide specific phospholipase C (PI-PLC) has been one of the most extensively studied. Several isoforms of PI-PLCs have been identified in the nucleus, namely PI-PLC-b1, c1, d1 and f; however, the b1 isozyme is the best characterized. In the present review, we focus on the signal transduction-related metabolism of nuclear PI-PLC and review the most convincing evidence for PI-PLC expression and activity being involved in differentiation and proliferation programmes in several cell systems. Moreover, nuclear PI-PLC is an intermediate effector and interactor for nuclear inositide signalling. The inositide cycle exists and shows a biological role inside the nucleus. It is an autonomous lipid-dependent signalling system, independently regulated with respect to the one at the plasma membrane counterpart, and is involved in cell cycle progression and differentiation.
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.
Molecular & Cellular Proteomics, 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 PKCI 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 PKCI could phosphorylate Ser 53 of the eEF1A2 isoform and that the association between eEF1A2 and PKCI was dependent on the phosphorylation status of eEF1A2.
Journal of Cellular Physiology, 2004
In the present work, we have analyzed the expression and subcellular localization of all the memb... more In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCbeta) family in muscle differentiation, given that nuclear PLCbeta1 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 PLCbeta1, beta2, beta3, beta4 have been assessed. As further characterization, we investigated the localization of PLCbeta isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCbeta4 was the most expressed isoform in the cytoplasm, whereas PLCbeta1 and beta3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCbeta1 isoform was expressed at the highest extent. A marked decrease of PLCbeta4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCbeta3 at both cytoplasmatic and nuclear level, whilst PLCbeta2 expression was almost undetectable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCbeta1 and the decrease of cytoplasmatic PLCbeta4, 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 PLCbetas.
Journal of Cellular Physiology, 2010
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'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.
Journal of Cellular Physiology, 2003
Phospholipase C β1 (PLCβ1) signaling in both cell proliferation and differentiation has been larg... more 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. ...
Italian journal of anatomy and embryology, 2017
Italian journal of anatomy and embryology, 2015
Italian journal of anatomy and embryology, 2016
The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characteri... more The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections. The evidence of a clinical correlation between the presence of a monoallelic gene deletion of Phospholipase Cβ1 (PLCβ1) and the progression of MDS to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments. Patients affected by MDS with a higher risk of AML evolution have a reduction in the expression of the nuclear PLCβ1, which is also epigenetically relevant in MDS. This strengthens the importance of PLCβ1 localization. In fact, PLCβ1 is a molecular target for hypomethylating agents, such Azacitidine (AZA)(1). High-risk MDS patients that respond to the drug showed an increased expression of nuclear PLCβ1 and its downstream target Cyclin D3 (CCND3), an induction of normal myeloid differentiation, and a better prognosis. Stemming from these data, ...
Handbook of Experimental Pharmacology, 2019
Nuclear inositides have a specific subcellular distribution that is linked to specific functions;... more 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.
Journal of Lipid Research, 2018
Nuclear phosphoinositide-specific phospholipases C (PI-PLCs) are a group of enzymes that hydrolyz... more Nuclear phosphoinositide-specific phospholipases C (PI-PLCs) are a group of enzymes that hydrolyze phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] to inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) that, in turn, are key second messengers involved in the activation of several signaling pathways related to many critical cellular functions (1, 2). Indeed, nuclear PI-PLCs play pivotal roles in cell cycle regulation (3, 4), cell proliferation, cell differentiation, membrane trafficking, and gene expression (5, 6). Recent evidence on the role of nuclear PI-PLC signaling in cell cycle regulation and cell proliferation paved the way to new fields of research related to different strategic physiopathological mechanisms in many cellular systems and diseases. This is the case of PI-PLC1, which plays a role in the physiopathology of brain disorders (along with PI-PLC1), hematological malignancies, and neuromuscular diseases, but also of PI-PLC-, which has been associated with fertility disorders. All in all, the imbalance of nuclear PI-PLC isoenzymes, such as PI-PLC1 and PI-PLC-, can lead to pathology (7).
Advances in biological regulation, 2017
Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the geneti... more Phosphoinositide-phospholipase C-β1 (PLC-β1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation and osteogenesis. During myogenic differentiation of murine C2C12 myoblasts, PLC-β1 signaling pathway involves the Inositol Polyphosphate Multikinase (IPMK) and β-catenin as downstream effectors. By means of c-jun binding to cyclin D3 promoter, the activation of PLC-β1 pathway determines cyclin D3 accumulation. However, osteogenesis requires PLC-β1 expression and up-regulation but it does not affect cyclin D3 levels, suggesting that the two processes require the activation of different mediators.
Italian journal of anatomy and embryology, 2012
Oncotarget, Jan 30, 2014
PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin ... more PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCβ1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCβ1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCβ1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCβ1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCβ1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effect...
Advances in biological regulation, 2013
Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older ... more 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. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia.
Advances in biological regulation, 2012
International Journal of Molecular Medicine, 2006
Phosphoinositide-specific phospholipase C (PI-PLC) ß1 is a key enzyme in nuclear signal transduct... more Phosphoinositide-specific phospholipase C (PI-PLC) ß1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLCß1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLCß1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLCß1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCß1. To analyze and quantify the levels of the two different splicing variants of PI-PLCß1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PI-PLCß1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLCß1b mRNA compared to PI-PLCß1a mRNA. Our data support the contention that the deletion of the PI-PLCß1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the 1a isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.
FEBS Journal, 2013
Evidence accumulated over the past 20 years has highlighted the presence of an autonomous nuclear... more Evidence accumulated over the past 20 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signalling molecules are important components of signalling pathways operating within the nucleus. Nuclear polyphosphoinositide (PI) signalling relies on the synthesis and metabolism of phosphatidylinositol 4,5-bisphosphate, which can modulate the activity of effector proteins and is a substrate of signalling enzymes. 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. Among the nuclear enzymes involved in PI metabolism, inositide specific phospholipase C (PI-PLC) has been one of the most extensively studied. Several isoforms of PI-PLCs have been identified in the nucleus, namely PI-PLC-b1, c1, d1 and f; however, the b1 isozyme is the best characterized. In the present review, we focus on the signal transduction-related metabolism of nuclear PI-PLC and review the most convincing evidence for PI-PLC expression and activity being involved in differentiation and proliferation programmes in several cell systems. Moreover, nuclear PI-PLC is an intermediate effector and interactor for nuclear inositide signalling. The inositide cycle exists and shows a biological role inside the nucleus. It is an autonomous lipid-dependent signalling system, independently regulated with respect to the one at the plasma membrane counterpart, and is involved in cell cycle progression and differentiation.
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.
Molecular & Cellular Proteomics, 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 PKCI 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 PKCI could phosphorylate Ser 53 of the eEF1A2 isoform and that the association between eEF1A2 and PKCI was dependent on the phosphorylation status of eEF1A2.
Journal of Cellular Physiology, 2004
In the present work, we have analyzed the expression and subcellular localization of all the memb... more In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCbeta) family in muscle differentiation, given that nuclear PLCbeta1 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 PLCbeta1, beta2, beta3, beta4 have been assessed. As further characterization, we investigated the localization of PLCbeta isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCbeta4 was the most expressed isoform in the cytoplasm, whereas PLCbeta1 and beta3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCbeta1 isoform was expressed at the highest extent. A marked decrease of PLCbeta4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCbeta3 at both cytoplasmatic and nuclear level, whilst PLCbeta2 expression was almost undetectable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCbeta1 and the decrease of cytoplasmatic PLCbeta4, 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 PLCbetas.
Journal of Cellular Physiology, 2010
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'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.
Journal of Cellular Physiology, 2003
Phospholipase C β1 (PLCβ1) signaling in both cell proliferation and differentiation has been larg... more 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. ...