Forebrain-specific ablation of phospholipase Cγ1 causes manic-like behavior.

Manic episodes are one of the major diagnostic symptoms in a spectrum of neuropsychiatric disorders that include schizophrenia, obsessive-compulsive disorder and bipolar disorder (BD). Despite a possible association between BD and the gene encoding phospholipase Cγ1 (PLCG1), its etiological basis remains unclear. Here, we report that mice lacking phospholipase Cγ1 (PLCγ1) in the forebrain (Plcg1f/f; CaMKII) exhibit hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, hyperhedonia, hyperphagia, impaired learning and memory and exaggerated startle responses. Inhibitory transmission in hippocampal pyramidal neurons and striatal dopamine receptor D1-expressing neurons of Plcg1-deficient mice was significantly reduced. The decrease in inhibitory transmission is likely due to a reduced number of γ-aminobutyric acid (GABA)-ergic boutons, which may result from impaired localization and/or stabilization of postsynaptic CaMKII (Ca2+/calmodulin-dependent protein kinase II) at inhibitory synapses. Moreover, mutant mice display impaired brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent synaptic plasticity in the hippocampus, which could account for deficits of spatial memory. Lithium and valproate, the drugs presently used to treat mania associated with BD, rescued the hyperactive phenotypes of Plcg1f/f; CaMKII mice. These findings provide evidence that PLCγ1 is critical for synaptic function and plasticity and that the loss of PLCγ1 from the forebrain results in manic-like behavior.

Studies of inositol phospholipid-specific phospholipase C.

Inositol phospholipid-specific phospholipase C is the enzyme that generates phosphoinositide-derived messenger molecules. Mammalian cells contain at least five immunologically distinct phospholipase C enzymes that appear to be separate gene products. Complete amino acid sequences of four of these isozymes have been established. The overall sequence similarity is surprisingly low for enzymes catalyzing the same chemical reaction: three of them show limited amino acid sequence similarity to each other in two narrow regions, and the fourth enzyme is completely different. The diversity in primary structure together with different regional and cellular expression of the isozymes suggests that each isozyme has a defined function in processing the physiological response of different cell types to a variety of external stimuli and that each is regulated differently.

Requirement for the L-type Ca(2+) channel alpha(1D) subunit in postnatal pancreatic beta cell generation.

Pancreatic beta cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of alpha(1D) gene-knockout (alpha(1D)(-/-)) mice show that the L-type calcium channel, alpha(1D), is required for proper beta cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated alpha(1D)(-/-) islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, alpha(1C). Histologically, newborn alpha(1D)(-/-) mice had an equivalent number of islets to wild-type mice. In contrast, adult alpha(1D)(-/-) mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in beta cell generation. TUNEL staining showed that there was no increase in cell death in alpha(1D)(-/-) islets, and a 5-bromo-2' deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of beta cells in alpha(1D)(-/-) islets.

Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I.

It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) beta1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC beta1. In cells overexpressing a PLC beta1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I.

Overexpression of phospholipase C-gamma 1 in familial adenomatous polyposis.

Phosphoinositide-specific phospholipase C (PLC) isozymes occupy a central role in the signal transduction system by regulating various cellular processes including proliferation and differentiation. In the present study, we examined the contents of PLCs in colorectal adenomas, carcinomas, and normal mucosa obtained from 4 familial adenomatous polyposis patients to find out whether this enzyme plays any role in the pathogenesis of adenomas and/or carcinomas in familial adenomatous polyposis. Radioimmunoassay and immunoblot analysis revealed that in contrast to little difference in PLC-beta 1 and PLC-delta 1 content, a considerably higher level of PLC-gamma 1 was detected in 3 of 4 cases for adenoma and in all cases for carcinoma as compared to normal mucosa. The level of PLC-gamma 1 expression increased from normal mucosa to adenoma, and finally to carcinoma progressively. Immunohistochemical findings also confirmed this observation. Likewise, activity of PLC-gamma 1 was considerably higher in adenomas and carcinomas than in normal mucosa. These results suggest that PLC-gamma 1-mediated signal transduction may play a significant role in the progression of colorectal tumors in patients with familial adenomatous polyposis.

Overexpression of phospholipase C-gamma1 in rat 3Y1 fibroblast cells leads to malignant transformation.

Phospholipase C-gamma1 (PLC-gamma1) mediates signals from various extracellular origins to evoke cellular events such as mitogenesis. Previously, we reported that PLC-gamma1 was highly expressed in colorectal cancer and familial adenomatous polyposis, suggesting that PLC-gamma1 might be oncogenic. In this study, we have established rat 3Y1 fibroblasts that overexpress whole PLC-gamma1 and src homology 2 (SH2)-SH2-SH3 domain of PLC-gamma1. These cells showed a transformed phenotype and were tumorigenic when transplanted into nude mice. These results indicate that overexpression of PLC-gamma1 could transform rat fibroblasts, and the transformation is mediated by SH2-SH2-SH3 domain of PLC-gamma1.

Inhibition of the EGF-induced activation of phospholipase C-gamma1 by a single chain antibody fragment.

Phospholipase C-gamma1(PLC-gamma1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-gamma1-specific inhibitors are commonly employed to investigate the mechanism of the PLC-gamma1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-gamma1 with high affinity (K(d)=1.9x10(-8) M) in vitro. F7-scFv also bound to PLC-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing sequestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.

Immunological identification of cholesterol ester hydrolase in the steroidogenic tissues, adrenal glands and testis.

Monoclonal antibodies were generated against the purified pancreatic cholesterol ester hydrolase (CEH, EC 3.1.1.13) to examine the expression of CEH in various bovine tissues. The presence of CEH isozyme antigenically indistinguishable from pancreatic enzyme in the steroidogenic tissues, adrenal glands and testis has been first demonstrated here using the immunoprecipitation method. These results suggest that CEH isozyme, similar to pancreatic CEH, might be involved in the cholesterol metabolism in the steroidogenic tissue.

Localization of two forms of phospholipase C-beta1, a and b, in C6Bu-1 cells.

Phospholipase C-beta1 (PLC-beta1), one of the PLC-beta isozymes, exists as two immunologically distinguishable polypeptides of 150 (PLC-beta1a) and 140 kDa (PLC-beta1b) which are encoded in two distinct transcripts and generated by alternative splicing of a single gene. In this study, the subcellular localization of the two phospholipases C-beta1 proteins was examined in rat C6Bu-1 glioma cells using immunological techniques. Immunoblot analysis revealed that the two forms of PLC-beta1 were detectable in both cytosolic and nuclear fractions. PLC-beta1a appeared to be located preferentially in the cytosol, whereas PLC-beta1b was found predominantly in the nuclei of C6Bu-1 cells. Immunocytochemical experiments confirmed the differential localization of the two PLC-beta1 species in C6Bu-1 cells. These results suggest that the two PLC-beta1 proteins may have different physiological roles in the cell.

Phorbol myristate acetate-dependent association of protein kinase C alpha with phospholipase D1 in intact cells.

A phospholipase D1 (PLD1) was purified from rat brain by the use of antibody-coupled protein A Sepharose. We found that protein kinase C alp (PKCalpha) stimulated PLD1 activity in the presence of phorbol myristate acetate (PMA). PMA-dependent association of PKCalpha with PLD1 was verified in NIH-3T3 fibroblast cells, and COS7 cells transiently expressing PLD1 as well as in vitro suggesting that the activation of PLD1 resulted from direct association of PKCalpha with PLD1.

Phospholipase D1 is located and activated by protein kinase C alpha in the plasma membrane in 3Y1 fibroblast cell.

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKC alpha translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKC alpha was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKC alpha in 3Y1 cells.

Trp-Lys-Tyr-Met-Val-D-Met is a chemoattractant for human phagocytic cells.

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling.

Hydrogen peroxide-induced phospholipase D2 activation in lymphocytic leukemic L1210 cells.

Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholipase D (PLD). However, it was still unclear how this activation occurs and what the molecular identity of the H2O2-stimulated PLD isozyme is. This study shows that H2O2 potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD2. In addition, H2O2 increased PLD activity only in PLD2-transfected COS-7 cells and not in PLD1-transfected cells. This suggests that PLD2 is selectively activated by H2O2. Depletion of extracellular Ca2+ with EGTA completely blocked the H2O2-induced PLD activation, indicating that Ca2+ influx is required. Moreover, pretreatment of the cells with the protein kinase C (PKC) inhibitors GF-109203X and RO-31-8220 and down-regulation of PKCalpha by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate inhibited the H2O2-stimulated PLD2 activity, which points to the involvement of PKCalpha. Based on these new findings we suggest that PLD2 activity is specifically up-regulated by H2O2 and that the H2O2-induced PLD2 activation is mediated by Ca2+ influx and PKCalpha activation.

Caspase-mediated cleavage of TRAF3 in FasL-stimulated Jurkat-T cells.

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) proteins play a central role in the early steps of signal transduction by TNFR superfamily proteins, which induce various cellular responses, including apoptosis. Influences of TRAF proteins on the regulation of cell death and physical interactions between TRAFs and caspases have been reported. In this study, we demonstrate that TRAF3 is proteolyzed during cell death in a caspase-dependent manner. TRAF3 was found to be cleaved by incubation with caspase3 in vitro and by Fas- or CD3-triggering in Jurkat-T cells. The Fas- or CD3-induced cleavage of TRAF3 was blocked by caspase inhibitors and by introduction of alanine substitutions for D347 and D367 residues. Furthermore, the amino-terminal fragment of TRAF3 showed a different intracellular localization from the full-length TRAF3 with preferential distribution to particulate fractions and the nucleus. These findings suggest that TRAF3 may be regulated by caspases during apoptosis of T cells.

Trp-Lys-Tyr-Met-Val-D-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes.

Among the phagocytic leukocytes, monocytes have the important role of clearing out parasitic microorganisms. They accomplish this through production of toxic metabolites of oxygen. Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), a peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes, including monocytes, binds to a unique cell surface receptor and stimulates superoxide generation, killing of Staphylococcus aureus, and activation of phospholipase D (PLD) in human monocytes. Preincubation of the cells with a PI-specific phospholipase C (PLC) inhibitor (U-73122), protein kinase C inhibitor (GF109203X), or intracellular Ca2+ chelator (BAPTA/AM) before the peptide stimulus totally inhibits the peptide-induced PLD activation and superoxide generation. On the other hand, tyrosine kinase inhibitor genistein only partially inhibits the peptide-induced processes. The peptide-induced bacteria killing activity shares regulatory mechanisms for PLD activation with the superoxide generation, which is inhibited in the presence of 1-butanol. We suggest that the peptide stimulates PLD downstream of PLC activation and PLD activation in turn is essential for the peptide-induced immunological functions such as the superoxide generation and killing of bacteria by human monocytes.

Identification of the peptides that inhibit the stimulation of thyrotropin receptor by Graves' immunoglobulin G from peptide libraries.

Graves' disease is characterized by the overproduction of thyroid hormones due to the persistent stimulation of TSH receptor by autoantibodies. To determine the epitopes recognized by the autoantibodies, an enzyme-linked immunosorbent assay was developed that uses the human TSH receptor extracellular domain attached to plastic wells. The total IgG from some of the Graves' patients interacted with the bound TSH receptor (TSHR) at a significantly higher level than that in normal individuals. The IgG preparation that showed the highest binding activity was used for the identification of peptide sequences that prevent binding of Graves' IgG to TSHR from positional scanning synthetic peptide combinatorial libraries. A hexapeptide mixture, X1X2FDDA (X1 is a mixture of E, M, and Y; X2 is a mixture of E, H, and T), was found to be effective for inhibiting the binding of Graves' IgG to the TSHR. Further fractionation of X1X2FDDA showed that the following three sequences were highly effective: EEFDDA, ETFDDA, and EHFDDA. The second position of the three peptides did not appear to be important. The peptides also inhibited the cAMP synthesis induced by IgG of four of eight patients with Graves' disease tested. The synthesis of cAMP by TSH was also inhibited by the peptides to some extent. The peptide sequences most likely mimic a part of the conformational epitopes recognized by at least one class of Graves' IgG.

A G-protein-coupled 130 kDa phospholipase C isozyme, PLC-beta 4, from the particulate fraction of bovine cerebellum.

A 130 kDa PLC isozyme was purified from the particulate fraction of bovine cerebellum. This PLC was recognized by a polyclonal antiserum generated against the purified 97 kDa PLC-beta 4. Reconstitution of the purified 130 kDa PLC with the membranes of C6 Bu-1 cells in the presence of GTP gamma S or AlF4- resulted in PLC activation as well as the association of PLC with the membranes. Both the association and activation were revoked when the membrane was washed with 2 M KCl. The 97 kDa PLC-beta 4 did not associate with membranes. These data suggest that the 130 kDa PLC is the intact form of PLC-beta 4 the activity of which is likely to be regulated by a G-protein on the membrane.

Transformation of rat fibroblasts by phospholipase C-gamma1 overexpression is accompanied by tyrosine dephosphorylation of paxillin.

We previously have shown that the overexpression of phospholipase C-gamma1 (PLC-gamma1) in rat 3Y1 fibroblasts results in malignant transformation (Chang, J.-S., Noh, D.Y., Park, I.A., Kim, M;.J., Song, H., Ryu, S.H. and Suh, P.-G. (1997) Cancer Res. 57, 5465-5468). The transformed cells, which initially are in an elongated and flat form after seeding in plastic dishes, become rounded during continued culture. We found that tyrosine dephosphorylation of paxillin accompanies this morphological change of the transformed cells and that PLC-gamma1 co-immunoprecipitates together with paxillin and vice versa, but not after the cells have become round. Transformed cells growing on fibronectin-pre-coated dishes regain their flat morphology and this is accompanied by paxillin tyrosine phosphorylation. Furthermore, immunoprecipitation analysis showed that paxillin forms a heteromeric complex with PLC-gamma1 in cells grown on fibronectin. These results suggest that a complex formation between paxillin and PLC-gamma1 may play a role in cell-substrate adhesion.

Localization of phospholipase C-gamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation.

Upon epidermal growth factor treatment, phospholipase C-gamma1 (PLC-gamma1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC-gamma1 and its tyrosine phosphorylation are localized in caveolae by caveolin-enriched low-density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl-beta-cyclodextrin (MbetaCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC-gamma1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MbetaCD shows no effect on tyrosine phosphorylation level of PLC-gamma1. Our findings suggest that, for proper signaling, PLC-gamma1 phosphorylation has to occur at PtdInsP(2)-enriched sites.

Proteolytic cleavage of epidermal growth factor receptor by caspases.

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.