Phosphoinositide-specific Phospholipase C Beta Research Articles

Nuclear Inositide Signalling: Role of PI‐PLCbeta1 in MDS and AML

The nuclear signaling evoked by phosphoinositide‐ specific‐phospholipase C (PI‐PLC)beta1 targets specific regions of Cyclin D3 as well CD24 promoter, hinting at a role in haematopoiesis, given that both Cyclin D3 and CD24 are involved in the very early steps of this process. PI‐PLCbeta1 could also play a role in the progression of Myelodysplastic Syndromes (MDS) into Acute Myeloid Leukemia (AML) since the mono‐allelic deletion of PI‐PLC beta1 is responsible in MDS patients for a higher risk of evolution to AML. Given that a single patient treated with azacitidine, a DNA methyltransferase inhibitor, displayed a direct correlation between PI‐PLCbeta1 gene expression and drug responsiveness, we hypothesized that PI‐PLCbeta1 could be a target for demethylating therapy. By quantifying the degree of PI‐PLCbeta1 methylation and gene expression in MDS patients at baseline and during azacitidine administration we found an increase of PI‐PLCbeta1 mRNA in responder patients and a reduction of promoter methylation. The molecular response correlated to and anticipated the clinical one, suggesting that PI‐PLCbeta1 gene reactivation predicts azacitidine responsiveness. Our results demonstrate for the first time that PI‐PLCbeta1 promoter is hypermethylated in high‐risk MDS patients and that the amount of PI‐PLCbeta1 mRNA predicts the clinical response to azacitidine.

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.

Subnuclear localization and differentiation‐dependent increased expression of DGK‐ζ in C2C12 mouse myoblasts

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-zeta might play some fundamental role during myogenic differentiation of C2C12 cells.

Phosphoinositide-specific phospholipase Cbeta1 expression is not linked to nerve growth factor-induced differentiation, cell survival or cell cycle control in PC12 rat pheocromocytoma cells.

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.

Subcellular distribution of phospholipase C isoforms in rodent pancreas and gastric mucosa.

Phosphoinositide-specific phospholipase C (PLC) has been implicated as a participant in cell proliferation as well as enzyme and hormone secretion. Defining the subcellular distribution of PLC isoforms would possibly contribute to further understanding of their function. We investigated the intracellular distribution of four PLCs (beta1, beta2, beta3, and gamma1) in mouse pancreatic cells as well as mouse and rat gastric mucosa cells by ultrastructural immunocytochemistry. In pancreatic acinar cells, PLCbeta1 and PLCgamma1 were demonstrated in the zymogen granules while PLCbeta2 was present in the granulae as well as the endoplasmic reticulum (ER), and PLCbeta3 was prominent in the ER. In the endocrine pancreas, PLCbeta2 immunolabeling was expressed in the secretory granulae of alpha, beta, delta, and pancreatic polypeptide cells. PLCbeta3 showed a slight labeling in the nucleus and ER of all four pancreatic endocrine cell types while PLCgamma1 was prominent in alpha cell granulae. In the gastric mucosa cells, PLCbeta2 was highly expressed in the heterochromatin areas and in the ER of parietal, chief, mucous, and enterochromaffin-like cells. PLCbeta3 were expressed in a manner similar to PLCbeta2 in those cells; however, no immunoreaction was seen in the ER of parietal cell. PLCgamma1 was demonstrated in the chief cell granulae. One possible, although yet speculative, interpretation of our results is that the studied PLC isoforms may be involved in processing in pancreatic secretory granulae and that nuclear PLCbeta2 and PLCbeta3 signaling pathways may be operative in the cells of the gastric mucosa.

Cell-specific signaling and structure-activity relations of parathyroid hormone analogs in mouse kidney cells.

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cbeta (PLCbeta). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCbeta. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1-31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3-34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7-34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1-34) and PTH-(1-31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCbeta. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.

G protein‐independent stimulation of human myocardial phospholipase C by mastoparan

1 Phosphoinositide-specific phospholipase C (PLC) is involved in the regulation of many cellular functions. In the myocardium, PLC-generated second messengers play a role in the regulation of contractile function and in the pathophysiology of myocardial hypertrophy. 2 In the present study, the effect of mastoparan, a tetradecapeptide which is capable of activating heterotrimeric G proteins by mimicking the action of an activated receptor, on membrane-bound human myocardial PLC, was investigated in a cell-free assay with exogenous phospholipids as a substrate. 3 Mastoparan stimulated human myocardial PLC approximately two fold with a half-maximal effect at approximately 2 microM and a maximal effect at 10 microM. The peptide did not alter the dependence of PLC on free calcium ions. In order to exclude non-specific effects of mastoparan due to its amphiphilic properties, different mastoparan derivatives were used as positive and negative controls. Mas17, an inactive mastoparan analogue with physical properties very similar to mastoparan, did not induce substantial PLC stimulation in human myocardial membranes. In contrast, Mas7, the most active mastoparan derivative known, caused a more pronounced PLC activation compared with the mother compound indicating that the effect was sequence-specific. Human myocardial PLC stimulation was pertussis toxin-insensitive and could not be abolished by addition of excess alpha-subunits from purified retinal transducin or by excess GDP or GDP/beta S. In order to investigate whether mastoparan stimulate PLC via pertussis toxin-insensitive alpha q, a deletion mutant of PLC beta 2 deficient of the site of interaction with alpha q-subunits was expressed in COS-1 cells. Both wild-type and mutant PLC beta 2 were similarly sensitive to stimulation by mastoparan. It is concluded that mastoparan stimulates human myocardial PLC by a mechanism distinct from heterotrimeric G proteins.

Identification of three phospholipase C isotypes expressed in rat olfactory epithelium.

The expression of three phosphoinositide-specific phospholipase C (PLC) isotypes in rat olfactory epithelium was investigated using monoclonal antibodies. In intact animals, PLC beta 1 was not expressed in the olfactory epithelium but was found in glands below the epithelium. However, following unilateral olfactory bulbectomy (OBX), PLC beta 1 was expressed in the dentrites of some olfactory receptor neurons, primarily in the endoturbinates on the unoperated side. PLC gamma 1 immunoreactivity was found in the apices of sustentacular cells and in glands below the epithelium. PLC delta 1 immunoreactivity was found in the glands and in the perinuclear region and dendrites of some receptor neurons. Since none of the PLC isotypes studied were expressed in the cilia of receptor neurons, the results suggest that another PLC isotype is likely to be involved in mediating olfactory transduction.

Phospholipase C activity in cerebrospinal fluid following subarachnoid hemorrhage related to brain damage.

Phosphoinositide-specific phospholipase C (PLC) activities were measured in CSF from patients after subarachnoid hemorrhage (SAH). Their PLC activities were significantly higher than those in control CSF. Moreover, there was an obvious correlation between the PLC activity in CSF collected on day 3 and the preoperative clinical grade. The PLC activity was also closely correlated with the level of neuron-specific enolase as a marker of brain damage. Furthermore, the PLC activities were partially purified from CSF of patients after SAH and were immunologically identified to be PLC beta, PLC gamma, and PLC delta. These results suggest that PLCs are released into the CSF from brain tissue in conjunction with the initial hemorrhage and that their activity may reflect the extent of brain damage.

Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes.

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.

Mineral fiber-mediated activation of phosphoinositide-specific phospholipase c in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells

Given the role of phosphoinositide-specific phospholipase C (PLC) isozymes in the control of cell growth and differentiation we were prompted to analyze the expression of some of these PLC in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells. The effects of several fluoro-edenite fibers were compared with those of tremolite, a member of the calcic amphibole group of asbestos that originates from Calabria (Italy), and crocidolite, that, due to its high toxicity, is one of the most studied asbestos amphiboles. Our data show an increased expression of both PLC beta1 and PLC gamma1 in A549 cells treated with asbestos-like fibers, hinting at a role of PLC signalling in those cancerous cells.

Identification of a novel cytosolic poly-phosphoinositide-specific phospholipase C (PLC-86) as the major G-protein-regulated enzyme.

Activation of phosphoinositide-specific phospholipase C (PLC) generates two intracellular signals which play major roles in many cellular processes including secretion, proliferation and contraction. PLC activation by many receptors occurs via a guanine nucleotide regulatory protein, Gp. PLCs are found predominantly in the cytosolic fraction though some activity is membrane-associated. At least four families of iso-enzymes of PLC (alpha, beta, gamma and delta) have been identified, but there is only scant evidence to indicate that any of the mammalian cytosolic activities are involved in G-protein-regulated signalling. In this study we demonstrate that the PLC activity from rat brain cytosol can be regulated in a G-protein-dependent manner in a reconstituted system using pre-permeabilized HL60 cells. We identify two enzymes, PLC-beta and a novel 86 kDa enzyme (designated PLC-epsilon) as the G-protein-regulated enzymes. PLC-epsilon was found to be the major G-protein-regulated enzyme.