Phosphoinositide-specific Phospholipase C Research Articles

Arabidopsis Ca2+-dependent protein kinase CPK3 mediates relationship of putative inositol triphosphate receptor with slow-type anion channel

It has been suggested in Arabidopsis thaliana (L.) Heynh. cv. Columbia that, contrary to 30 μM abscisic acid (ABA), 20 μM ABA induces guard cell Ca2+ mobilization through activating phosphoinositide-specific phospholipase C (PI-PLC)-dependent inositol 1,4,5-triphosphate (IP3) production. Here, it was investigated whether Ca2+-dependent protein kinase, CPK3 or CPK6 would mediate ABA-induced stomatal closure downstream of IP3 production. In the knockout cpk3-1 mutant, the PLC inhibitor (U73122) adjusted 20 μM ABA-induced stomatal closure to the extent observed in the knockout cpk6-1 and cpk3-1cpk6-1 mutants and the wild type, whereas, in the wild type, the inhibitor of IP3-induced Ca2+ mobilization, xestospongin C (XeC), adjusted this closure to the extent observed in the cpk3-1 mutant. The Ca2+ buffer, EGTA and XeC positively interacted with the slow anion channel blocker, anthracene-9-carboxylic acid (9-AC) to inhibit 20 μM ABA-induced stomatal closure, which was suppressed in the dexamethasone-inducible AtPLC1 antisense transgene or the knockout cpk3-1, cpk6-1, cpk3-1cpk6-1 and NADPH oxidase atrbohD/F mutants. Discrete concentrations of 9-AC or another slow anion channel blocker (probenecid) negatively interacted with the Ca2+ buffer, BAPTA or the inhibitor of cyclic ADP-ribose-induced Ca2+ mobilization, ruthenium red, to inhibit 30 μM ABAinduced stomatal closure in the wild type but not in the cpk6-1, cpk3-1cpk6-1 and atrbohD/F mutants. Based on so far revealed features of the tested compounds and plant materials, interpretation of the results confirmed that guard cell ABA concentration discriminates between two Ca2+ mediations and outlined that one of them sequentially implicates CPK6, PLC1, a putative IP3 receptor homologue, CPK3, and the slow anion channel, whereas the other one excludes AtPLC1-dependent IP3 production and CPK3.

Genetic and transcriptional analysis of phosphoinositide-specific phospholipase C in Plasmodium

Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled.

Phospholipase Cɛ has a crucial role in ultraviolet B-induced neutrophil-associated skin inflammation by regulating the expression of CXCL1/KC

Phospholipase C (PLC) ɛ is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCɛ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCɛ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCɛ+/+) and PLCɛ gene knockout (PLCɛ−/−) mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m2 on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCɛ+/+ mice, irradiation with 1 and 2.5 kJ/m2 UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m2 of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCɛ−/− mice, UVB (1 or 2.5 kJ/m2)-induced neutrophil infiltration was markedly suppressed compared with PLCɛ+/+ mice. The suppression of neutrophil infiltration in PLCɛ−/− mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCɛ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCɛ−/− mice. These results indicate that PLCɛ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.

Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation.

Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP2 and the production of the secondary messenger IP3 in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca2+ signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP2/IP3 probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca2+ and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.

Role of Phosphoinositide-Specific Phospholipase C η2 in Isolated and Syndromic Mental Retardation

Deletions in the distal region of the short arm of chromosome 1 (1p36) are widely diffuse, both as somatic abnormalities in tumors and as constitutive in the congenital 1p36 deletion syndrome. The deletion size varies from 1.5 to 10 Mb, with common breakpoints located from 1p36.13 to 1p36.33. Patients bearing constitutional deletion of a smaller region, 1p36.3, present with a number of features, including mental retardation. The gene PLCH2, codifying for the phosphoinositide-specific phospholipase C (PI-PLC) η2, maps on the 1p36.32 region. PI-PLC η2, expressed in the brain after birth, is a key enzyme in cellular calcium mobilization. In the brain, calcium plays a role in axon growth and retraction, growth cone guidance, synapse formation, and responses to various neurotransmitters. For its role in the nervous system, PI-PLC η2 might be a putative candidate gene for the neurodevelopmental delay observed in patients bearing 1p36.3 deletions.

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Phospholipase Cε as a potential molecular target for anti-inflammatory therapy and cancer prevention

Phospholipase Ce (PLCe), encoded by PLCE1, is the phosphoinositide-specific PLC regulated by the ras proto-oncogene product Ras and its relative Rap1. Like other PLC isoforms, PLCe catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. To understand the physiological function of PLCe, we have created and analyzed genetically-modified mice in which PLCe is inactivated or overproduced. PLCe knockout (KO) mice are resistant to two-stage skin chemical carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene and promoted by phorbol 12-myristate 13-acetate (PMA) and to intestinal tumorigenesis caused by the loss-of-function mutation of the APC tumor suppressor gene. In PLCe KO mice, inflammation, such as that induced by a single application of PMA, that associated with tumorigenesis, and that in the elicitation phase of allergic contact hypersensitivity, is also attenuated as compared to that in wild-type mice. Conversely, overexpression of PLCe in the epidermis results in the development of skin inflammation that partly shares the features with human psoriasis. In this article, we summarize the data showing the role of PLCe in tumorigenesis and inflammation and discuss the future directions of the study of PLCe for the development of therapies to control inflammation and to prevent cancer progression.

Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.

Involvement of PI-PLCβ1 in insulin secretion in MIN6 pancreatic β-cells

Diabetes is a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long-term damage, dysfunction and failure of various organs (WHO 1999). Its prevalence worldwide was 171,000,000 in 2000 and it is estimated 366,000,000 in 2030. Failure of pancreatic β-cells in insulin secretion plays a major pathogenic role in the emergence of type 2 diabetes mellitus. Inositol lipid metabolism is involved in complex signalling pathways that regulate the stimulus-secretion coupling processes. In particular, evidences indicate that the activation of phosphoinositide-specific phospholipase C (PI-PLC) and the resulting hydrolysis of phosphatidylinositol-4,5bisphosphate [PtdIns(4,5)P2] in diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are critical to several events such as opening of Ca 2+ stores, mediating the effect of many hormones and neurotransmitters and regulating gene expression. We used murine MIN6 pancreatic β-cells to investigate the role of PI-PLCβ1 in insulin secretion. Analysis of the subcellular distribution revealed that PI-PLCβ1 localized mainly in the nuclei, as observed by immunocytochemical analysis at TEM and by western blot. PI-PLCβ1 is involved in basal insulin secretion, since PI-PLCβ1 silencing, by means of siRNAs, resulted in a decreased level of insulin secretion at low glucose dose. We have performed PCR array profiling to identify signal transduction pathways involved in insulin secretion, regulated by PI-PLCβ1. From these studies many genes resulted upor downregulated when PI-PLCβ1 in silenced, and brought to interest on PI-PLCβ1 signal transduction in β-cells.

α1‐Adrenoceptor‐mediated depletion of phosphatidylinositol 4, 5‐bisphosphate inhibits activation of volume‐regulated anion channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE Volume‐regulated anion channels (VRACs) play an important role in cell‐volume regulation. α1‐Adrenoceptor stimulation by phenylephrine (PE) suppressed the hypotonic activation of VRAC current in mouse ventricular cells and regulatory volume decrease (RVD) was also absent in PE‐treated cells. We examined whether the effects of α1‐adrenoceptor stimuli on VRAC current were modulated by phosphatidylinositol signalling.EXPERIMENTAL APPROACH Whole‐cell patch‐clamp method was used to record the hypotonicity‐induced VRAC current in mouse ventricular cells. RVD was analyzed by videomicroscopic measurement of cell images.KEY RESULTS The attenuation of VRAC current by PE was suppressed by α1A‐adrenoceptor antagonists (prazosin and WB‐4101), anti‐Gq protein antibody and a specific phosphoinositide‐specific phospholipase C (PLC) inhibitor (U‐73122), but not by antagonists for α1B‐, α1D‐ or β‐adrenoceptor, or protein kinase C inhibitors. The inhibition of VRAC by PE was antagonized by intracellular excess phosphatidylinositol 4,5‐bisphosphate (PIP2), while intracellular anti‐PIP2 antibody (PIP2 Ab) inhibited the activation of VRAC currents. When cells were loaded with phosphatidylinositol 3,4,5‐trisphosphate (PIP3) with or without PIP2 Ab, PE little affected the VRAC current. Extracellular m‐3M3FBS (an activator of PLC) suppressed VRAC in the absence of PE, and this effect was reversed by intracellular excess PIP2.CONCLUSIONS AND IMPLICATIONS Our results indicate that the stimulation of α1A‐adrenoceptors by PE inhibited the activation of cardiac VRAC current via PIP3 depletion brought about by PLC‐dependent reduction of membrane PIP2 level.

Enhancement of ultraviolet B-induced skin tumor development in phospholipase Cε -knockout mice is associated with decreased cell death

Phospholipase C (PLC) ε is a phosphoinositide-specific PLC regulated by small guanosine triphosphatases including Ras and Rap. Our previous studies revealed that PLCε gene-knockout (PLCε(-/-)) mice exhibit marked resistance to tumor formation in two-stage skin chemical carcinogenesis using 7,12-dimethylbenz(a)anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. In this model, PLCε functions in tumor promotion through augmentation of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. In this study, we have further assessed the role of PLCε in tumorigenesis using a mouse model of ultraviolet (UV) B-induced skin tumor development. We irradiated PLCε(+/+), PLCε(+/-) or PLCε(-/-) mice with doses of UVB increasing from 1 to 10 kJ/m(2) three times a week for a total of 25 weeks and observed tumor formation for up to 50 weeks. In sharp contrast to the results from the two-stage chemical carcinogenesis study, PLCε(-/-) mice developed a large number of neoplasms including malignant tumors, whereas PLCε(+/+) and PLCε(+/-) mice developed a relatively small number of benign tumors. However, UVB-induced skin inflammation was greatly suppressed in PLCε(-/-) mice, as observed with 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, implying that PLCε's role in the suppression of UVB-induced tumorigenesis is not mediated by inflammation. Studies of the tumor initiation stage revealed that UVB-induced cell death in the skin was markedly suppressed in PLCε(-/-)mice. Our findings identify a novel function for PLCε as a critical molecule regulating UVB-induced cell death and suggest that resistance to UVB-induced cell death conferred by the absence of PLCε is closely related to the higher incidence of skin tumor formation.

The phospholipase C inhibitor U‐73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine). U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release. U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC.

Expression pattern and sub‐cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U‐73122 in rat astrocytoma cells

Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase

The photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca2+-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [32P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C4-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca2+-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK.

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.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

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.

Nuclear inositide signaling in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are defined as clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. Although distinct morphologic subgroups exist, the natural history of MDS is progression to acute myeloid leukemia (AML). However, the molecular the mechanisms the underlying MDS evolution to AML are not completely understood. Inositides are key cellular second messengers with well-established roles in signal transduction pathways, and nuclear metabolism elicited by phosphoinositide-specific phospholipase C (PI-PLC) beta1 and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Recent findings evidenced the role played by nuclear lipid signaling pathways, which could become promising therapeutic targets in MDS. This review will provide a concise and updated revision of the state of art on this topic.

Leishmania amazonensis: Heme stimulates (Na + + K +)ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways

In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na + + K +)ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50 nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH 3 and U73122, specific inhibitors of PI-PLC. (Na + + K +)ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50 nM. This effect was completely reversed by 10 nM calphostin C, an inhibitor of PKC. Thus, the effect of 50 nM heme on (Na + + K +)ATPase activity is completely abolished by ET-18-OCH 3 and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na + + K +)ATPase activity through a PI-PLC/PKC signaling pathway.

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.