Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic ß-cells by activating Phospholipase C signal pathway in diabetic mice.

In early stage of diabetes, insulin secretion from pancreatic ß-cells is increased to deal with the elevated blood glucose. Previous studies have reported that islet-produced carbon monoxide (CO) is associated with increased glucose-stimulated insulin secretion from ß-cells. However, this compensatory mechanism by which CO may act to enhance ß-cell function remain unclear. In this study, we revealed that CO promoted intracellular calcium ([Ca2+]i) elevation and glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells in leptin receptor deficient db/db mice but not in C57 mice. The stimulatory effects of CO on ß-cell function in db/db mice was blocked by inhibition of Phospholipase C (PLC) signaling pathway. We further demonstrated that CO triggered [Ca2+]i transients and enhanced GSIS in C57 islets when ß-cells overexpressed with PLCγ1 and PLCδ1, but not PLCß1. On the other hand, reducing PLCγ1 and PLCδ1 expressions in db/db islets dramatically attenuated the stimulatory effects of CO on ß-cell function, whereas interfering PLCß1 expression had no effects on CO-induced ß-cell function enhancement. Our findings showing that CO elevated [Ca2+]i and enhanced GSIS by activating PLC signaling through PLCγ1 and PLCδ1 isoforms in db/db pancreatic ß-cells may suggest an important mechanism by which CO promotes ß-cell function to prevent hyperglycemia. Our study may also provide new insights into the therapy for type II diabetes and offer a potential target for therapeutic applications of CO.

Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4).

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.

Ca2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP3), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipases C (PLCs), that leads to Ca2+ release from endoplasmic reticulum by InsP3 receptors (InsP3R). Ca2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca2+ levels have specific biological effects that differ from those of Ca2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16INK4A+ and p21Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.

Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines.

3-Amino-2-arylcarboxamide-thieno[2,3-b]pyridines are a known class of antiproliferative compounds with activity against the phospholipase C enzyme. To further investigate the structure activity relationships of these derivatives a series of analogues were prepared modifying key functional groups. It was determined that modification of the 3-amino and 2-aryl carboxamide functionalities resulted in complete elimination of activity, whilst modification at C-5 allowed compounds of greater activity to be prepared.

Phospholipase C δ1 in macrophages negatively regulates TLR4-induced proinflammatory cytokine production and Fcγ receptor-mediated phagocytosis.

Macrophages are key players in the innate immune response. Turnover of phosphoinositides (PI), particularly phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2), has been implicated in macrophage functions such as toll-like receptor (TLR)-mediated cytokine production and phagocytosis. However, PI metabolizing enzymes responsible for macrophage functions are not well defined. The phospholipase C (PLC) family of enzymes is critical in PI(4,5)P2 turnover. In this study, we investigated the role of PLCδ1, a prototype PLC, in macrophages on the expression of inflammation-associated genes and phagocytosis. Lipopolysaccharides (LPS) signal through TLR4 to produce proinflammatory cytokines such as interleukin (IL)-1ß. LPS stimulation of both RAW264.7 murine macrophages and murine bone marrow-derived macrophages resulted in lower PLCδ1 mRNA and protein expression levels, compared to that in the control. Using chemical inhibitor compounds, we demonstrated that the up-regulation of p38 MAPK activity led to down-regulation of PLCδ1 mRNA expression in macrophages. PLCδ1 reduction by RNAi or gene deletion resulted in greater LPS-induced IL-1ß expression than that observed in the control siRNA-treated cells, without increasing TLR4 cell surface expression. PLCδ1 also negatively regulated LPS-induced cell spreading. Analysis of Fcγ receptor-mediated phagocytosis demonstrated an increased phagocytosis index after PLCδ1 knockdown in RAW264.7 cells. Conversely, overexpression of PLCδ1 reduced phagocytosis whereas catalytic inactive PLCδ1 had no effect. Altered levels of PLCδ1 affected the binding of opsonized latex beads with cells, rather than the phagocytic activity. Taken together, the data suggest that PLCδ1 negatively regulates LPS-induced production of IL-1ß and Fcγ receptor-mediated phagocytosis in macrophages.

The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.

Increase of Ca(2+) influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca(2+) homeostasis as they are highly selective toward Ca(2+) and constitutively activated. Thus, we investigated the involvement of these channels in Ca(2+) influx in CF and non-CF human bronchial epithelial cell lines. 16HBE14o-, CFBE41o- cell lines, primary human airway epithelial cells (hAEC) and freshly isolated human airway epithelial cells from CF and non-CF individuals were used. We showed that both channels are expressed in CF and non-CF cells and constitutive Ca(2+) influx was significantly higher (85%) in cells from CF individuals compared to cells from non-CF ones. Using the selective inhibitor of TRPV6 channel SOR-C27 and a siRNA strategy, our results revealed that TRPV6 was mostly involved in the increase of Ca(2+) influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca(2+) influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca(2+) influx was increased by 33% with U73122 and this increase was largely reduced in the presence of SOR-C27. PLC inhibition in CF cells by U73122 had no effect on Ca(2+) influx. These results showed that PLC-PIP2 pathway is dysregulated in CF cells and leads to the increase of TRPV6 activity. The regulation of TRPV6 by PLC-PIP2 pathway implicates the specific PLC isoform, PLC-δ1. Immunoblot experiments revealed that expression of PLC-δ1 was decreased by 70% in CF cells. TRPV6 activity was normalized but not the level of expression of PLC-δ1 protein after F508del-CFTR rescue by low temperature for 48 h or treated for 24 h by 10 µM VX-809 in CF cells. This study revealed TRPV6 and PLC-δ1 as critical actor of Ca(2+) homeostasis in CF human bronchial epithelial cells.

Phospholipase C-δ(1) regulates interleukin-1ß and tumor necrosis factor-α mRNA expression.

Phospholipase C-δ(1) (PLCδ(1)) is a widely expressed highly active PLC isoform, modulated by Ca(2+) that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLCδ(1) modulated expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLCδ(1) was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLCδ(1) knockdown enhanced expression IL-1ß and tumor necrosis factor-α (TNF-α) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLCδ(1) knock down caused persistently high Nfκb levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLCδ(1) knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1ß and TNF-α mRNA's induced by PLCδ(1) knockdown. Our results show that loss of PLCδ(1) enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nfκb pathway. Our findings are consistent with the idea that PLCδ(1) is a suppressor of PKC activity.

p122 protein enhances intracellular calcium increase to acetylcholine: its possible role in the pathogenesis of coronary spastic angina.

OBJECTIVE: Phospholipase C-δ1 activity is enhanced in patients with coronary artery spasm, and a p122 protein was recently cloned to potentiate phospholipase C-δ1 activity. To investigate the role of p122 in enhanced vasomotility, we examined p122 expression in the cultured skin fibroblasts obtained from patients with and without coronary spasm, intracellular Ca(2+) concentration ([Ca(2+)]i) [corrected] at baseline and after stimulation with acetylcholine in the cells transfected with p122, and promoter in genomic DNA. METHODS AND RESULTS: [corrected] p122 protein and gene expression levels in patients with coronary spasm (n=11) were enhanced compared with levels in control subjects (n=9) (P<0.01 for both). [Ca(2+)](i) at baseline and the peak increase in [Ca(2+)](i) in response to acetylcholine were both 2 times higher in cells transfected with p122 than in those without p122. Conversely, knockdown of p122 resulted in diminished [Ca(2+)](i) response. In the p122 promoter analysis, the -228G/A and -1466C/T variants revealed the increase in luciferase activity. Although the -1466C/T variant was similar between 144 patients with coronary spasm and 148 controls, the -228G/A variant was more frequent in male patients than in male controls (P<0.05). CONCLUSIONS: The p122 protein is upregulated in patients with coronary spasm, causing increased [Ca(2+)](i) to acetylcholine, and thereby seems to be related to enhanced coronary vasomotility.

General and versatile autoinhibition of PLC isozymes.

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-beta2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-beta2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

Phospholipase C-delta1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1.

Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.

Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.