Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide

Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide‐specific phospholipase C (PI‐PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI‐PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI‐PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL‐6. PI‐PLCs expression in LPS treated neonatal rat astrocytes performed by using RT‐PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI‐PLC beta1, beta4 and gamma1 in all intervals analysed; PI‐PLC delta1 at 6, 18 and 24 h; PI‐PLC delta3 at 6 h after treatment. PI‐PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub‐cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI‐PLCs expression and distribution may play a role in ongoing inflammation process of CNS. J. Cell. Biochem. 109: 1006–1012, 2010. © 2010 Wiley‐Liss, Inc.     

Cell‐cycle‐dependent PC‐PLC regulation by APC/CCdc20‐mediated ubiquitin‐proteasome pathway

Phosphatidylcholine‐specific phospholipase C (PC‐PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC‐PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC‐PLC were co‐immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH‐7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC‐PLC and Cdc20 were co‐localized in the perinuclear endoplasmic reticulum region (the “juxtanuclear quality control” compartment, JUNQ). The expression level and activities of PC‐PLC changed in a cell‐cycle‐dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC‐PLC, and caused PC‐PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC‐PLC regulation in cell cycles is controlled by APC/C^Cdc20‐mediated UPP. J. Cell. Biochem. 107: 686–696, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.     

Staphylococcal phosphatidylinositol‐specific phospholipase C potentiates lung injury via complement sensitisation

Staphylococcus aureus is frequently isolated from patients with community‐acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol‐specific phospholipase C (PI‐PLC); however, the role of PI‐PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI‐PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol‐anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI‐PLC‐positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc‐isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild‐type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc^? mutant. Following treatment with cobra venom factor to deplete complement, the wild‐type strain with PI‐PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI‐PLC‐positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI‐PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.     

Functional roles of PC‐PLC and Cdc20 in the cell cycle,proliferation, and apoptosis

Phosphatidylcholine‐specific phospholipase C (PC‐PLC) is the major enzyme in the Phosphatidylcholine (PC) cycle and is involved in many long‐term cellular responses such as activation, proliferation, and differentiation events. Cell division cycle 20 homolog (Cdc20) is an essential cell‐cycle regulator required for the completion of mitosis. Our previous studies identified the interaction between PC‐PLC and Cdc20. Through the interaction, Cdc20 could mediate the degradation of PC‐PLC by Cdc20‐mediated ubiquitin proteasome pathway (UPP). In this study, we found that PC‐PLC might not be involved in cancer metastasis. Inhibition of PC‐PLC by D609 could cause cell proliferation inhibition and apoptosis inhibition in CBRH‐7919 cells. Inhibition of PC‐PLC could also influence the cell cycle by arresting the cells in G1 phase, and Cdc20 might be involved in these processes. Taken together, in this report, we provided new evidence for the functional roles of PC‐PLC and Cdc20 in the cell cycle, proliferation, and apoptosis in CBRH‐7919 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Thrombin stimulates RPE cell motility by PKC‐ζ‐ and NF‐κB‐dependent gene expression of MCP‐1 and CINC‐1/GRO chemokines

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial‐mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose‐dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR‐2 and CCR‐2 chemokine receptors. Whereas inhibition of CXCR2 by Sb‐225002 and of CCR2 by Rs‐504393 partially prevented hirudin‐sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro‐32‐0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC‐ζ pseudosubstrate and by the nuclear factor‐kappa B (NF‐κB) inhibitor BAY11‐7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK–ERK–PI3K‐mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC‐ζ. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

Phospholipase C‐η2 is required for retinoic acid‐stimulated neurite growth

Phospholipase C‐η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃‐sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)‐induced Neuro2A cell differentiation. PLCη2 expression increased two‐fold during a 4‐day differentiation period. Stable expression of PLCη2‐targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA‐treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two‐hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co‐localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA‐induced phosphorylation of LIMK1 and cAMP‐responsive element‐binding protein was reduced in PLCη2 knock‐down cells. The phosphoinositide‐binding properties of the PLCη2 PH domain, assessed using a FRET‐based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co‐localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.     

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

Plant phosphatidylinositol‐specific phospholipase C at the center of plant innate immunity

Understanding plant resistance to pathogenic microbes requires detailed information on the molecular mechanisms controlling the execution of plant innate immune responses. A growing body of evidence places phosphoinositide‐specific phospholipase C (PI‐PLC) enzymes immediately downstream of activated immune receptors, well upstream of the initiation of early defense responses. An increase of the cytoplasmic levels of free Ca²⁺, lowering of the intercellular pH and the oxidative burst are a few examples of such responses and these are regulated by PI‐PLCs. Consequently, PI‐PLC activation represents an early primary signaling switch between elicitation and response involving the controlled hydrolysis of essential signaling phospholipids, thereby simultaneously generating lipid and non‐lipid second messenger molecules required for a swift cellular defense response. Here, we elaborate on the signals generated by PI‐PLCs and their respective downstream effects, while providing an inventory of different types of evidence describing the involvement of PI‐PLCs in various aspects of plant immunity. We project the discussed information into a model describing the cellular events occurring after the activation of plant immune receptors. With this review we aim to provide new insights supporting future research on plant PI‐PLCs and the development of plants with improved resistance.     

Involvement of receptor tyrosine phosphatase DEP‐1 mediated PI3K‐cofilin signaling pathway in Sorafenib‐induced cytoskeletal rearrangement in hepatoma cells

Sorafenib is a multikinase inhibitor that has been reported to induce cell growth inhibition through the Raf‐MAPK signaling pathway. We now report that Sorafenib treatment of Hep3B and PLC/PRF/5 human hepatoma cells also results in morphological changes and cell detachment in culture. Actin cytoskeletal analysis of Sorafenib‐exposed Hep3B cells showed a loss of polymerized F‐actin and a concomitant increase in unpolymerized G‐actin, implying that Sorafenib‐induced cell shape changes may be related to actin cytoskeletal rearrangement by inhibiting actin polymerization. Cofilin, an actin depolymerization factor, was found to be dephosphorylated and thus activated by Sorafenib, consistent with the observed increase in unpolymerized G‐actin. In examining likely mechanisms, we found that Sorafenib induced activation of the cofilin phosphatase Slingshot 1 (SSH‐1), since endogenous SSH‐1 from Sorafenib‐treated Hep3B cells was able to dephosphorylate cofilin in a concentration dependent manner. The activation of SSH‐1 by Sorafenib is probably regulated by the PI3K pathway, since Sorafenib can induce PI3K and its substrate Akt phosphorylation, and both PI3K inhibitors Ly294002 and wortmannin antagonized Sorafenib‐mediated cofilin dephosphorylation. Furthermore, we found that Sorafenib induced c‐Met phosphorylation at Tyr‐1349 but not Tyr‐1234, which is probably mediated by inhibition of receptor tyrosine phosphatase density enhanced phosphatase‐1 (DEP‐1). Our data provide evidence that besides inhibition of the Raf‐MAPK pathway, Sorafenib might also regulate hepatoma cell growth via alteration of receptor‐mediated cytoskeletal rearrangement. J. Cell. Physiol. 224: 559–565, 2010. © 2010 Wiley‐Liss, Inc.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.     

Thrombin down‐regulates the TGF‐β‐mediated synthesis of collagen and fibronectin by human proximal tubule epithelial cells through the EPCR‐dependent activation of PAR‐1

Human proximal tubule (HK‐2) cells are commonly used as cellular models to understand the mechanism by which inflammatory mediators cause renal injury. It has been observed that thrombin stimulates the expression of TGF‐β, extracellular matrix (ECM) proteins and proinflammatory cytokines by HK‐2 cells. These in vitro responses correlate well with the pathology of glomerular and tubular diseases observed in acute renal injury. HK‐2 cells express PAR‐1 and the thrombin activation of this receptor has been reported to up‐regulate the TGF‐β‐mediated expression of ECM proteins, suggesting a possible pathogenic role for PAR‐1 signaling by thrombin in acute renal injury. On the other hand, several recent studies have indicated that activated protein C plays a renoprotective role, thus inhibiting the inflammatory responses and attenuating renal injury, presumably by activating the same cell surface receptor. In this study, we show that HK‐2 cells express endothelial protein C receptor (EPCR) and that the occupancy of this receptor by protein C switches the signaling specificity of thrombin so that the activation of PAR‐1 by thrombin inhibits the TNF‐α‐mediated synthesis of IL‐6 and IL‐8 and down‐regulates the TGF‐β‐mediated expression of ECM proteins. These results suggest a possible protective role for EPCR in acute kidney injury. J. Cell. Physiol. 225: 233–239, 2010. © 2010 Wiley‐Liss, Inc.     

Aquaporin Trafficking in Plant Cells: An Emerging Membrane‐Protein Model

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and small solutes across biological membranes. In plants, AQPs exhibit a high multiplicity of isoforms in relation to a high diversity of sub‐cellular localizations, at the plasma membrane (PM) and in various intracellular compartments. Some members also exhibit a dual localization in distinct cell compartments, whereas others show polarized or domain‐specific expression at the PM or tonoplast, respectively. A diversity of mechanisms controlling the routing of newly synthesized AQPs towards their destination membranes and involving diacidic motifs, phosphorylation or tetramer assembly is being uncovered. Recent approaches using single particle tracking, fluorescence correlation spectroscopy and fluorescence recovery after photobleaching have, in combination with pharmacological interference, stressed the peculiarities of AQP sub‐cellular dynamics in environmentally challenging conditions. A role for clathrin and sterol‐rich domains in cell surface dynamics and endocytosis of PM AQPs was uncovered. These recent advances provide deep insights into the cellular mechanisms of water transport regulation in plants. They also point to AQPs as an emerging model for studying the sub‐cellular dynamics of plant membrane proteins .     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

PKC isoenzymes differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation

Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.