BMP-2 induced expression of PLC beta1 that is a positive regulator of osteoblast differentiation

C2C12 is an immortalized mouse myoblast cell line. The cells readily proliferate in high-serum conditions, and differentiate and fuse in low-serum conditions. While this cell line is a very useful tool to study aspects of myogenesis, metabolism and muscle biology, however, treatment of C2C12 cells with bone morphogenic protein (BMPs) induces cells to differentiate into osteoblasts. Osteoblast differentiation is controlled by diversified signaling proteins and transcription factors, essentially BMP-2, Osterix (Osx/Sp7) and Runx2, finally associating with the expression of late osteoblast marker genes, like ALPL and Bglap. These peculiarities make C2C12 progenitor cells a skillful prototype to investigate the molecular mechanism that control cell destiny specification and terminal differentiation. In the current investigation, we took improvement of the differentiation peculiarities of the mouse C2C12 cell line to analyze whether changes in PLCbeta1 expression and its nuclear localization might regulate or affect their terminal osteogenic differentiation. We demonstrated that overexpression of PLCβ1 enhances the osteogenic differentiation of C2C12 elicited by BMP-2 as demonstrated by the presence of osteoblast marker genes expression. In the present study we also showed that miR-214 suppressed osteogenic differentiation through the regulation of nuclear PLCβ1 by targeting Osterix

PI3Kα-selective inhibitor alpelisib (BYL719), may be effective as anticancer agents in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer that arises from the skeletal muscle. Recent studies have identified an important role of AKT signaling in RMS progression. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the class I PI3K inhibitors [1] in human rhabdomyosarcoma cell lines (embryonal rhabdomyosarcoma RD and A204, alveolar rhabdomyosarcoma SJCRH30). We used a panel of four compounds which specifically target PI3K isoforms including the PI3Kα-selective (p110α ) inhibitor alpelisib BYL719, currently in clinical development by Novartis Oncology, the p110β TGX-221 inhibitor, the p110γ CZC24832, the p110δ CAL-101inhibitor and the dual p110α/p110δ inhibitor AZD8835. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTT assays, cell cycle by flow cytometry , apoptosis by caspase 3/7 assay and Western blot, as well as the phosphorylation status of the pathway. BYL719 treatment resulted in G1 phase cell cycle arrest and apoptosis. BYL719 administered in combination with CAL-101, for 48 h and 72h, decreased cell viability and induced apoptosis in a marked synergistic manner. Taken together, our findings indicate that BYL719, either alone or in combination with p110δ CAL-101inhibitor, may be an efficient treatment for human rhabdomyosarcoma cells that have aberrant upregulation of the PI3K signaling pathway for their proliferation and survival

Nuclear DGKα regulates cell cycle progression in K562 cells

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases (1). Phosphatidylinositols (PIs) play an important role in nuclear function regulation and behave differently from their counterparts in the cytoplasm. The autonomous nuclear PI cycle in eukaryotic cells is involved in different regulation processes, from cell proliferation to differentiation and many others (2). At nuclear level an array of kinases and phosphatases can modulate PIs. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol (DAG) and induce the synthesis of phosphatidic acid. We Investigated DGKα localization and function in human erythroleukemia cell line K562. Synchronization experiments at different cell cycle checkpoints showed an important expression of DGKα in the nuclear fraction of this cell model, slightly peaking at G2/M. This suggested that DGKα might have a function in nuclear signaling. In particular, nuclear DGKα expression can modulate cell cycle progression, leading to changes in the phosphorylated status of the Retinoblastoma protein (pRb), thus, regulating G1/S transition: DGKα silencing or downregulation leads to impaired G1/S transition and its overexpression leads to S phase progression. The molecular mechanism by which nuclear DGKα controls pRb phosphorylation and therefore cell cycle regulation in K562 cell line are still unclear. Further studies are needed to better understand the role of DGKα in relation to other pivotal PIs involved in cell cycle regulation in the hematopoietic system

Phospholipase c beta 1 (PLCb1) in acute myeloid leukemia (AML): a novel potential therapeutic target

Acute myeloid leukemia (AML) is the most common type of leukemia in adults in which leukemic myeloid derived cells replace normal blood cells leading to a loss in systemic function. Once initiated the disease develops rapidly and is typically fatal within weeks or months if left untreated. AML is a complex disease and although, the exact causes of the development of AML are unknown, risk factors include age, pre-leukemic diseases such as myelodysplastic syndrome, exposure to chemicals and radiation and genetics. The mainstay treatment is still chemotherapy together with stem cell replacement therapy and while life expectancy has increased slowly, the 5 year survival rates range between 12 and 70% with relapse rates as high as 70% depending on the subtype (canceruk). These statistics illustrate the urgent requirement for the development of novel targeted therapeutics. Phospholipases C (PLC) are critical intracellular signaling enzymes that control a wide range of cellular functions including proliferation and apoptosis that have been implicated in myelodysplastic diseases and in leukemia (Faenza et al., 2013; Shah et al.). Importantly they constitute a highly druggable family of enzymes distinct from other well established drug development targets such as protein kinases. Using the human leukemic cell line THP1, we carried out a small targeted RNAi screen to establish a role of all known PLCs in cell growth, differentiation and maintenance of the transformed phenotype. We discovered that silencing of PLCb1 or PLCH2 resulted in a strong growth arrest. PLCb1 knockdown also initiated apoptosis and attenuated growth of THP1 cells in semisolid culture, which is known to reflect the ability of cells to induce leukemia in vivo. Accordingly, we found that knockdown of PLCb1 strongly attenuated THP1-mediated development of leukemia in mice. These growth inhibitory effects of PLCb1 knockdown were extended to a mouse model of human leukaemia induced by the MLL-AF9 translocation and to human primary leukemia cells. Of direct importance to the consideration for drug development we observed that PLCb1 knockdown selectively attenuated the growth of primary human AML cells, without effecting cell growth and differentiation of normal CD34+ hematopoietic stem and progenitor cells from healthy donors. We therefore propose PLCb1 as a novel candidate for a therapeutic target in AML

Nuclear phospholipase C β1 signaling, epigenetics and treatments in MDS.

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.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. © 2012 Elsevier Ltd

PLC-beta 1 regulates the expression of miR-210 during mithramycin-mediated erythroid differentiation in K562 cells

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 effects were the same encountered in cells overexpressing PLCβ1. Therefore we suggest a novel role for PLCβ1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCβ1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression

Nuclear phospholipase Cβ1 interactome: a morphological and proteomic approach

Inositide-dependent phospholipase Cβ1 (PI-PLCβ1b) has two isoforms generated by alternative splicing (PI-PLCβ1a and PI-PLCβ1b). In murine erythroleukemia (MEL) cells both the isoforms are present within the nucleus, but PI-PLCβ1b is exclusively nuclear. Our group has demonstrated that PI-PLCβ1 nuclear localisation is crucial for its function, although the mechanism by which PI-PLCβ1 is imported into the nucleus has never been carefully investigated. The purpose of the present study was to get more insights on the protein interactome of PI-PLCβ1b, namely the proteins present in the nucleus. Immuno-affinity purification coupled with tandem mass spectrometry analysis have been used to purify and identify PI-PLCβ1b interaction binding partners from Friend’s erythroleukemia isolated nuclei. Gene ontology and protein-protein interaction network were performed to analyze data. Some interactions were already characterized, such as the binding with the splicing factor SRp20 and the lamin B. Among the proteins identified, the binding of eEF1A and prohibitin 2 with PI-PLCβ1b was confirmed by western blot analysis. Of particular interest was the identification of importin a, importin b1 and Ran, which interact with PI-PLCβ1b. These proteins are believed to be involved in the import mechanism from the cytoplasm to the nucleus. Further analysis by overexpressing both wild type and cytoplasmatic mutant of PI-PLCβ1, suggests that importin b1 is responsible for the localisation of PI-PLCβ1b in the nucleus, giving new insight into the mechanism of trafficking of this signaling molecule

Nuclear Phosphoinositides as Key Determinants of Nuclear Functions

Polyphosphoinositides (PPIns) are signalling messengers representing less than five per cent of the total phospholipid concentration within the cell. Despite their low concentration, these lipids are critical regulators of various cellular processes, including cell cycle, differentiation, gene transcription, apoptosis and motility. PPIns are generated by the phosphorylation of the inositol head group of phosphatidylinositol (PtdIns). Different pools of PPIns are found at distinct subcellular compartments, which are regulated by an array of kinases, phosphatases and phospholipases. Six of the seven PPIns species have been found in the nucleus, including the nuclear envelope, the nucleoplasm and the nucleolus. The identification and characterisation of PPIns interactor and effector proteins in the nucleus have led to increasing interest in the role of PPIns in nuclear signalling. However, the regulation and functions of PPIns in the nucleus are complex and are still being elucidated. This review summarises our current understanding of the localisation, biogenesis and physiological functions of the different PPIns species in the nucleus

Epigenetic regulation of nuclear PLCbeta1 and Cyclin D3 during Azacitidine treatment

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, our goal was to analyze the correlation between CCND3, PLCβ1 and AZA treatment. Firstly, we treated two different cellular lines, AML HL60 and histiocytic lymphoma U937, with AZA 5μM (Ec50 for HL60 cells) for 24 hours. Then, we used Real-Time PCR and Western blot to quantify both gene and protein expression. Moreover, we showed that CCND3 promoter has one CpG island. For this reason, it is possible that AZA could directly affect both PLCβ1 and CCND3 promoters. Therefore, we studied PLCβ1 binding to CCND3 promoter by chromatin immunoprecipitation (CHIP), before and after AZA treatment. Our results evidenced that the recruitment of PLCβ1 to CCND3 promoter is specifically increased after AZA treatment, leading to suppose that PLCβ1 could have a pivotal role in MDS with either a direct or indirect effect on cell cycle, proliferation and differentiation. These complicate relations need future deepening in order to demonstrate how PLCβ1 binding actually regulates CCND3 expression and how much this expression depends on CCND3 direct promoter demethylation and PLCβ1 control