PIP4Ks impact on PI3K, FOXP3, and UHRF1 signaling and modulate human regulatory T cell proliferation and immunosuppressive activity.

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2 They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

Emerging Roles of Phospholipase C Beta Isozymes as Potential Biomarkers in Cardiac Disorders.

Phospholipase C (PLC) enzymes represent crucial participants in the plasma membrane of mammalian cells, including the cardiac sarcolemmal (SL) membrane of cardiomyocytes. They are responsible for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into 1,2-diacylglycerol (DAG) and inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), both essential lipid mediators. These second messengers regulate the intracellular calcium (Ca2+) concentration, which activates signal transduction cascades involved in the regulation of cardiomyocyte activity. Of note, emerging evidence suggests that changes in cardiomyocytes' phospholipid profiles are associated with an increased occurrence of cardiovascular diseases, but the underlying mechanisms are still poorly understood. This review aims to provide a comprehensive overview of the significant impact of PLC on the cardiovascular system, encompassing both physiological and pathological conditions. Specifically, it focuses on the relevance of PLCß isoforms as potential cardiac biomarkers, due to their implications for pathological disorders, such as cardiac hypertrophy, diabetic cardiomyopathy, and myocardial ischemia/reperfusion injury. Gaining a deeper understanding of the mechanisms underlying PLCß activation and regulation is crucial for unraveling the complex signaling networks involved in healthy and diseased myocardium. Ultimately, this knowledge holds significant promise for advancing the development of potential therapeutic strategies that can effectively target and address cardiac disorders by focusing on the PLCß subfamily.

PIP4K2B: Coupling GTP Sensing to PtdIns5P Levels to Regulate Tumorigenesis.

Although guanine nucleotides are essential for cell growth, how their levels are sensed in mammalian cells is unknown. Sumita et al. show that PIP4K2B, a phosphoinositide kinase, is a molecular sensor that transduces changes in GTP into changes in the levels of the phosphoinositide PtdIns5P to modulate tumour cell growth.

Inositide-Dependent Nuclear Signalling in Health and Disease.

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.

Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders.

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCß1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCß1 and PI-PLCÉ£1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCß1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCß1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.

Phosphatidylinositol 5 Phosphate (PI5P): From Behind the Scenes to the Front (Nuclear) Stage.

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.

Phosphoinositide 3 Kinase Signaling in Human Stem Cells from Reprogramming to Differentiation: A Tale in Cytoplasmic and Nuclear Compartments.

Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.

Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.

PIP4K and the role of nuclear phosphoinositides in tumour suppression.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated lipid kinases that phosphorylate PtdIns5P to generate PtdIns(4,5)P2. There are three isoforms of PIP4Ks: PIP4K2A, 2B and 2C, which localise to different subcellular compartments with the PIP4K2B isoform being localised predominantly in the nucleus. Suppression of PIP4K expression selectively prevents tumour cell growth in vitro and prevents tumour development in mice that have lost the tumour suppressor p53. p53 is lost or mutated in over 70% of all human tumours. These studies suggest that inhibition of PIP4K signalling constitutes a novel anti-cancer therapeutic target. In this review we will discuss the role of PIP4K in tumour suppression and speculate on how PIP4K modulates nuclear phosphoinositides (PPIns) and how this might impact on nuclear functions to regulate cell growth. This article is part of a Special Issue entitled Phosphoinositides.

Deciphering signaling pathways in hematopoietic stem cells: the molecular complexity of Myelodysplastic Syndromes (MDS) and leukemic progression.

Myelodysplastic Syndromes, a heterogeneous group of hematological disorders, are characterized by abnormalities in phosphoinositide-dependent signaling, epigenetic regulators, apoptosis, and cytokine interactions within the bone marrow microenvironment, contributing to disease pathogenesis and neoplastic growth. Comprehensive knowledge of these pathways is crucial for the development of innovative therapies that aim to restore normal apoptosis and improve patient outcomes.

Nuclear PI-PLC ß1 and Myelodysplastic syndromes: from bench to clinics.

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. A number of MDS progresses to acute myeloid leukemia (AML) with the involvement of genetic and epigenetic mechanisms affecting PI-PLC ß1. The molecular mechanisms underlying the MDS evolution to AML are still unclear, even though it is now clear that the nuclear signaling elicited by PI-PLC ß1, Cyclin D3, and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Moreover, a correlation between other PI-PLCs, such as PI-PLC ß3, kinases and phosphatases has been postulated in MDS pathogenesis. Here, we review the findings hinting at the role of nuclear lipid signaling pathways in MDS, which could become promising therapeutic targets.

Nuclear PLCs affect insulin secretion by targeting PPARγ in pancreatic ß cells.

Type 2 diabetes is a heterogeneous disorder caused by concomitant impairment of insulin secretion by pancreatic ß 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 ß cells, PLCß1 localized in both nucleus and cytoplasm, PLCδ4 in the nucleus, and PLCγ1 in the 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-γ (PPARγ), a nuclear receptor transcription factor that regulates genes critical to ß-cell maintenance and functions. Silencing of PLCß1 and PLCδ4 resulted in a decrease in the PPARγ mRNA level. By means of a PPARγ-promoter-luciferase assay, the decrease could be attributed to a PLC action on the PPARγ-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 PPARγ as a novel molecular target of nuclear PLCs.

A role for PLCß1 in myotonic dystrophies type 1 and 2.

Phosphoinositide-phospholipase C ß1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. We previously demonstrated that nuclear PLCß1 activates the cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLCß1 is essential for cyclin D3 promoter activation and gene transcription, through c-jun/AP1. Myotonic dystrophy (DM) is the most prevalent form of muscular dystrophy in adults. DM type 1 (DM1) and type 2 (DM2) are dominantly inherited multisystem disorders. DM1 is triggered by the pathological expansion of a (CTG)(n) triplet repeat in the gene coding for DMPK, the dystrophia myotonica-protein kinase, whereas a (CCTG)(n) tetranucleotide repeat expansion in the ZNF9 gene, encoding a CCHC-type zinc finger protein, causes DM2. We found that, unlike in normal myotubes, the level of expression of PLCß1 in DM1 and DM2 cells was already elevated in proliferating cells. Treatment with insulin induced a dramatic decrease in the amount of PLCß1. During differentiation, cyclin D3 and myogenin were elevated in normal myotubes, whereas differentiating DM1 and DM2 cells did not increase these proteins. Forced expression of PLCß1 in DM1 and DM2 cells increased the expression of differentiation markers, myogenin and cyclin D3, and enhanced fusion of DM myoblasts. These results highlight again that PLCß1 expression is a key player in myoblast differentiation, functioning as a positive regulator in the correction of delayed differentiation of skeletal muscle in DM human myoblasts.

Nuclear phosphoinositides: location, regulation and function.

Lipid signalling in human disease is an important field of investigation and stems from the fact that phosphoinositide signalling has been implicated in the control of nearly all the important cellular pathways including metabolism, cell cycle control, membrane trafficking, apoptosis and neuronal conduction. A distinct nuclear inositide signalling metabolism has been identified, thus defining a new role for inositides in the nucleus, which are now considered essential co-factors for several nuclear processes, including DNA repair, transcription regulation, and RNA dynamics. Deregulation of phoshoinositide metabolism within the nuclear compartment may contribute to disease progression in several disorders, such as chronic inflammation, cancer, metabolic, and degenerative syndromes. In order to utilize these very druggable pathways for human benefit there is a need to identify how nuclear inositides are regulated specifically within this compartment and what downstream nuclear effectors process and integrate inositide signalling cascades in order to specifically control nuclear function. Here we describe some of the facets of nuclear inositide metabolism with a focus on their relationship to cell cycle control and differentiation.

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.

eEF1A phosphorylation in the nucleus of insulin-stimulated C2C12 myoblasts: Ser5³ is a novel substrate for protein kinase C ßI.

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 1α (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the cPKC isoforms, only PKCßI was demonstrated to be expressed in the nucleus of C2C12 myocytes and to co-immunoprecipitate with eEF1A. In-depth analysis using site-directed mutagenesis revealed that PKCßI could phosphorylate Ser5³ of the eEF1A2 isoform and that the association between eEF1A2 and PKCßI was dependent on the phosphorylation status of eEF1A2.

Roles of PI3K/AKT/mTOR Axis in Arteriovenous Fistula.

Renal failure is a worldwide disease with a continuously increasing prevalence and involving a rising need for long-term treatment, mainly by haemodialysis. Arteriovenous fistula (AVF) is the favourite type of vascular access for haemodialysis; however, the lasting success of this therapy depends on its maturation, which is directly influenced by many concomitant processes such as vein wall thickening or inflammation. Understanding the molecular mechanisms that drive AVF maturation and failure can highlight new or combinatorial drugs for more personalized therapy. In this review we analysed the relevance of critical enzymes such as PI3K, AKT and mTOR in processes such as wall thickening remodelling, immune system activation and inflammation reduction. We focused on these enzymes due to their involvement in the modulation of numerous cellular activities such as proliferation, differentiation and motility, and their impairment is related to many diseases such as cancer, metabolic syndrome and neurodegenerative disorders. In addition, these enzymes are highly druggable targets, with several inhibitors already being used in patient treatment for cancer and with encouraging results for AVF. Finally, we delineate how these enzymes may be targeted to control specific aspects of AVF in an effort to propose a more specialized therapy with fewer side effects.

The physiology and pathology of inositide signaling in the nucleus.

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.

Nuclear phospholipase C in biological control and cancer.

Inositol lipids are key regulators of several cellular functions. The identification of an independent nuclear polyphosphoinositides signaling machinery has led the way to find new roles for these molecules. PI-PLC-ß1 is the most extensively studied PLC isoform in the nuclear compartment and a key player in the regulation of nuclear lipid signaling. Nuclear PI-PLC-ß1 is involved in cell cycle progression and differentiation in response to growth factor stimulation. A growing body of evidence has demonstrated that nuclear phosphoinositides are also involved in cancer cell generation, proliferation, and resistance to apoptosis. Evidence on ex vivo human cancer cells from patients with myelodysplastic syndromes (MDS) confirmed these observations, suggesting the involvement of PI-PLC-ß1 both in the pathogenesis of the disease and in the progression of MDS to acute myeloid leukemia. These studies have offered new targets for the development of novel therapeutic strategies as well as new prognostic tools.

"Modulating Phosphoinositide Profiles as a Roadmap for Treatment in Acute Myeloid Leukemia".

Polyphosphoinositides (PPIns) and their modulating enzymes are involved in regulating many important cellular functions including proliferation, differentiation or gene expression, and their deregulation is involved in human diseases such as metabolic syndromes, neurodegenerative disorders and cancer, including Acute Myeloid Leukemia (AML). Given that PPIns regulating enzymes are highly druggable targets, several studies have recently highlighted the potential of targeting them in AML. For instance many inhibitors targeting the PI3K pathway are in various stages of clinical development and more recently other novel enzymes such as PIP4K2A have been implicated as AML targets. PPIns have distinct subcellular organelle profiles, in part driven by the specific localisation of enzymes that metabolise them. In particular, in the nucleus, PPIns are regulated in response to various extracellular and intracellular pathways and interact with specific nuclear proteins to control epigenetic cell state. While AML does not normally manifest with as many mutations as other cancers, it does appear in large part to be a disease of dysregulation of epigenetic signalling and many novel therapeutics are aimed at reprogramming AML cells toward a differentiated cell state or to one that is responsive to alternative successful but limited AML therapies such as ATRA. Here, we propose that by combining bioinformatic analysis with inhibition of PPIns pathways, especially within the nucleus, we might discover new combination therapies aimed at reprogramming transcriptional output to attenuate uncontrolled AML cell growth. Furthermore, we outline how different part of a PPIns signalling unit might be targeted to control selective outputs that might engender more specific and therefore less toxic inhibitory outcomes.