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In early stage of diabetes, insulin secretion from pancreatic β-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 β-cells. However, this compensatory mechanism by which CO may act to enhance β-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 β-cells in leptin receptor deficient db/db mice but not in C57 mice. The stimulatory effects of CO on β-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 β-cells overexpressed with PLCγ1 and PLCδ1, but not PLCβ1. On the other hand, reducing PLCγ1 and PLCδ1 expressions in db/db islets dramatically attenuated the stimulatory effects of CO on β-cell function, whereas interfering PLCβ1 expression had no effects on CO-induced β-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 β-cells may suggest an important mechanism by which CO promotes β-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.

Pilomatricoma, a benign skin appendage tumor, also known as calcifying epithelioma, consists of islands of epithelial cells histologically that contain anucleated cells in the center surrounded by basophilic cells and partial calcification. Sporadic pilomatricomas commonly have somatic mutations in the gene CTNNB1, but causative genes from germline and the underlying pathophysiology are unclear. In this study, we identified a germline missense variant of PLCD1 encoding PLCδ1, c.1186G>A (p.Glu396Lys), in a large Chinese family with autosomal dominant multiple pilomatricomas. Phospholipase C, a key enzyme playing critical roles in intracellular signal transduction, is essential for epidermal barrier integrity. The p.Glu396Lys variant increased the enzymatic activity of PLCδ1, leading to protein kinase C/protein kinase D/extracellular signal–regulated kinase1/2 pathway activation and TPRV6 channel closure, which not only resulted in excessive proliferation of keratinocytes in vitro and in vivo but also induced local accumulation of calcium in the pilomatricoma-like tumor that developed spontaneously in the skin of Plcd1E396K/E396K mice. Our results implicate this p.Glu396Lys variant of PLCD1 from germline leading to gain-of-function of PLCδ1 as a causative genetic defect in familial multiple pilomatricomas.

Pleckstrin homology (PH) domains are common modules of ∼120 amino acids found in proteins involved in signalling, cytoskeletal organization, membrane transport, and modification of phospholipids. Previous live cell studies have involved the use of the green-fluorescent protein (GFP) labelling of PH-domain of phospholipase C δ1 (PLC δ1) to study the interactions of molecules at the membrane interface.For this study, the aim was to construct and express the GFP-PH domain of PLC δ1 in the Saccharomyces cerevisiae BY4741. The transformants expressing GFP-PH domain of PLC δ1 displayed localised fluorescence to the cell periphery (plasma membrane) while the negative control expressed GFP within the cytoplasm only. No GFP was observed in the non-transformed yeast cells.Thus, this technique could be useful in future molecular interactions studies targeted specifically at the yeast cell membrane interface in live yeast cells.

Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.

Hereditary leukonychia (HL) is a rare nail dystrophy disease, and several different clinical manifestations and mutations in the phospholipase C δ 1 (PLCD1) gene have been reported. The present study reports on one Chinese family and one sporadic case of with HL. The family members exhibited an autosomal dominant pattern of inheritance with the involvement of all the fingers and toenails in all the patients. Of interest, most of the affected members had koilonychia during their childhood. Thus, the present study first used gene mapping with an aim to identify the pathogenic gene underlying koilonychia. Through genome-wide linkage analysis, the pathogenic area of koilonychia was identified on chromosome 3 with multipoint Log of Odds scores >2. A novel pathogenic mutation c.1384G>A (p.E462K) was identified in the PLCD1 gene in all the patients in the family, which confirmed the diagnosis of hereditary leukonychia. A novel mutation c.770G>A (p.R257H) was also detected in one sporadic case of leukonychia. On the basis of these findings and of previous studies, it is suggested that hereditary leukonychia may initially present as koilonychia, whereas hereditary koilonychia does not progress to leukonychia. Moreover, the present study identified two pathogenic variants of the PLCD1 associated with hereditary leukonychia, and highlights the significance of genetic diagnosis.

Background: MUC2, a major component of the mucus layer in the intestine, is associated with antimicrobial activity and gut immune system function. Currently, mucin is mainly known for its critical function in defense against toxic molecules and pathogens. In this study, we investigated the stimulatory effects of exogenous nicotinamide adenine dinucleotide (NAD+) on the expression of MUC2 in LS 174T goblet cells. Methods: Genes related to MUC2 synthesis were measured by quantitative real-time PCR (qPCR). To analyze the gene expression profiles of NAD+-treated LS 174T goblet cells, RNA sequencing was performed. MUC2 expression in the cells and secreted MUC2 were measured by immunocytochemistry (ICC) and ELISA, respectively. Results: NAD+ significantly stimulated MUC2 expression at mRNA and protein levels and increased the secretion of MUC2. Through RNA sequencing, we found that the expression of genes involved in arachidonic acid metabolism increased in NAD+-treated cells compared with the negative control cells. NAD+ treatment increased phospholipase C (PLC)-&delta, and prostaglandin E synthase (PTGES) expression, which was inhibited by the appropriate inhibitors. Among the protein kinase C (PKC) isozymes, PKC-&delta, was involved in the increase in MUC2 expression. In addition, extracellular signal-regulated kinase (ERK)1/2 and cyclic AMP (cAMP) response element-binding protein (CREB) transcript levels were higher in NAD+-treated cells than in the negative control cells, and the enhanced levels of phosphorylated CREB augmented MUC2 expression. Conclusions: Exogenous NAD+ increases MUC2 expression by stimulating the PLC-&delta, /PTGES/PKC-&delta, /ERK/CREB signaling pathway.

Glucocorticoids exert important therapeutic effects on airway smooth muscle (ASM), yet few direct targets of glucocorticoid signaling in ASM have been definitively identified. Here, we show that the transcription factor, Krüppel-like factor 15 (KLF15), is directly induced by glucocorticoids in primary human ASM, and that KLF15 represses ASM hypertrophy. We integrated transcriptome data from KLF15 overexpression with genome-wide analysis of RNA polymerase (RNAP) II and glucocorticoid receptor (GR) occupancy to identify phospholipase C delta 1 as both a KLF15-regulated gene and a novel repressor of ASM hypertrophy. Our chromatin immunoprecipitation sequencing data also allowed us to establish numerous direct transcriptional targets of GR in ASM. Genes with inducible GR occupancy and putative antiinflammatory properties included IRS2, APPL2, RAMP1, and MFGE8. Surprisingly, we also observed GR occupancy in the absence of supplemental ligand, including robust GR binding peaks within the IL11 and LIF loci. Detection of antibody-GR complexes at these areas was abrogated by dexamethasone treatment in association with reduced RNA polymerase II occupancy, suggesting that noncanonical pathways contribute to cytokine repression by glucocorticoids in ASM. Through defining GR interactions with chromatin on a genome-wide basis in ASM, our data also provide an important resource for future studies of GR in this therapeutically relevant cell type.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently mutated in CRC, and KRAS mutations promote cell motility, growth, and survival. We previously revealed that the expression of phospholipase C (PLC) δ1, one of the most basal PLCs, is down-regulated in colon adenocarcinoma, and that the KRAS signaling pathway suppresses PLCδ1 expression. Although recent studies revealed that KRAS mutations activate autophagy in cancer cells, a relation between PLCδ1 and autophagy remains unclear. Here, we found that PLCδ1 overexpression suppresses the formation of autophagosomes, which are key structures of autophagy, whereas endogenous PLCδ1 knockdown increases autophagosome formation in CRC cells. We also showed that PLCδ1 overexpression promotes cell death under nutrient deprivation. Furthermore, PLCδ1 overexpression suppresses the autophagy induced by the anti-cancer drug oxaliplatin and promotes cell death under oxaliplatin treatment. These data suggest that PLCδ1 negatively regulates autophagy, and PLCδ1 suppression contributes to the tolerance of CRC cells harboring KRAS mutations to nutrient deprivation and anti-cancer drug treatment.

Keratinocytes undergo a unique type of programmed cell death known as cornification, which leads to the formation of the stratum corneum (SC), the main physical barrier of the epidermis. A defective epidermal barrier is a hallmark of the two most common inflammatory skin disorders, psoriasis, and atopic dermatitis. However, the detailed molecular mechanisms of skin barrier formation are not yet fully understood. Here, we showed that downregulation of phospholipase C (PLC) δ1, a Ca2+-mobilizing and phosphoinositide-metabolizing enzyme abundantly expressed in the epidermis, impairs the barrier functions of the SC. PLCδ1 downregulation also impairs localization of tight junction proteins. Loss of PLCδ1 leads to a decrease in intracellular Ca2+ concentrations and nuclear factor of activated T cells activity, along with hyperactivation of p38 mitogen-activated protein kinase (MAPK) and inactivation of RhoA. Treatment with a p38 MAPK inhibitor reverses the barrier defects caused by PLCδ1 downregulation. Interestingly, this treatment also attenuates psoriasis-like skin inflammation in imiquimod-treated mice. These findings demonstrate that PLCδ1 is essential for epidermal barrier integrity. This study also suggests a possible link between PLCδ1 downregulation, p38 MAPK hyperactivation, and barrier defects in psoriasis-like skin inflammation.

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.

Calcium (Ca(2+)) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca(2+) 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)P(2)) within the nucleus, leading to Ca(2+) release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P(2) 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 Ca(2+) signaling and downstream events. We found that EGF-induced Ca(2+) signals are inhibited when translocation of EGFR is impaired. Nuclear Ca(2+) signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP(3)) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P(2) 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 InsP(3) 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.

Hereditary leukonychia is a rare genetic nail disorder characterized by distinctive whitening of the nail plate of all 20 nails. Hereditary leukonychia may exist as an isolated feature, or in simultaneous occurrence with other cutaneous or systemic pathologies. Associations between hereditary leukonychia and mutations in the gene encoding phospholipase C delta-1 (PLC?1) have previously been identified. However, the molecular mechanisms underlying PLC1 mutations and hereditary leukonychia remain uncharacterized. In the present study, we introduced hereditary leukonychia-linked human PLC?1 mutations (C209R, A574T and S740R) into equivalent residues of rat PLC1 (C188R, A553T and S719R), and investigated their effect on the biophysical and biochemical properties of the PLC1 protein. Our data suggest that these PLC1 mutations associated with hereditary leukonychia do not uniformly alter the enzymatic ability of this protein leading to loss/gain of function, but result in significantly divergent enzymatic properties. We demonstrate here for the first time the importance of PLC-mediated calcium (Ca2+) signalling within the manifestation of hereditary leukonychia. PLC?1 is almost ubiquitous in mammalian cells, which may explain why hereditary leukonychia manifests in association with other systemic pathologies relating to keratin expression. We thank Matilda Katan (University College London, UK) for the rat PLC?1 clone, and Xuexun Fang (Laboratory of Molecular Enzymology, Jilin University, China) for the pHSIE vector. MN was supported by a Marie Curie Intra-European Fellowship Award (628634). Funding for AT and J. Kashir was provided by the THALES ?Education and Lifelong Learning? Program No. 380170 (European Social Fund/Greek Government) and a Health Fellowship Award (National Institute for Social Care and Health Research, HP-14-16) respectively. Scopus

Background Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs. Methods Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493–2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation. Results PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice. Conclusions In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don’t display completely hairless phenotype as expected. Electronic supplementary material The online version of this article (10.1186/s12967-018-1512-9) contains supplementary material, which is available to authorized users.

Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein–coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif–containing proteins., This work was supported in part by National Institutes of Health Grants R01GM108733 and R01GM130120, American Cancer Society Grant RSG-13-362-01-TBE (to M. G.-M.), and by Medical Research Council Grant MR/M013944/1 (to G. S. B.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

The aim of the present study was to analyse the effect of PLCD4, PECR, FN1 and PNKD mutations on pig productive traits and tested the usefulness of targeted enrichment DNA sequencing method as tool for preselection of genetic markers. The potential genetic markers for pig productive traits were identified by using targeted enrichment DNA sequencing of chromosome 15 region that is QTL-rich. The selected mutations were genotyped by using HRM, Sanger sequencing and PCR-ACRS methods. The association study was performed by using GLM model in SAS program and included over 500 pigs of 5 populations maintained in Poland. The variation (C/T) of PLCD4 gene affected feed conversion, intramuscular fat and water exudation. The PNKD mutations were associated with texture parameters measured after cooking. In turn, the variation rs792423408 (C/T) in the FN1 gene influenced toughness measured in semimembranosus muscle and growth traits that was observed particularly in Duroc breed. Summarizing, the investigated gene variants delivered valuable information that could be used during developing SNP microarray for genomic estimated breeding value procedure in pigs. Moreover, the study showed that the TEDNA-seq method could be used to preselect the molecular markers associated with pig traits. However, the further association study that included large number animal populations is necessary. Electronic supplementary material The online version of this article (10.1007/s11033-018-4170-3) contains supplementary material, which is available to authorized users.

The genetic status of candidate tumor suppressor genes (TSGs) at chromosome 3p has not yet been elucidated in intrahepatic cholangiocarcinoma (iCCA). Herein, we retrospectively investigated 32 fresh iCCA case samples from a single medical institution to clarify mutations of 11 TSGs by next-generation sequencing. Validation of the mutations was performed on the MassARRAY platform or by high-resolution melting curve analysis. We then integrated the gene mutations into copy number alterations at chromosome 3p that had been generated in a previous study using the same fresh iCCA samples, and correlated the integration results with the clinicopathologic features. Nine of the 32 (28.1%) iCCA patients had gene mutations at chromosome 3p, totaling 11 mutations across five genes. Those included five (15.6%) BAP1 mutations, two each (6.3%) of CACNA2D3 and RASSF1 mutations, and one each (3.1%) of ATG7 and PLCD1 mutations. Six (18.8%) cases had concurrent loss of chromosome 3p and gene mutations. Patients with TSG mutations had shorter disease-free and survival times than those without the mutations. Our data showed that iCCA patients with TSG mutations at chromosome 3p faced an adverse prognosis. BAP1 was the common target of mutational inactivation and may be a principal driver of 3p21 losses.

Background Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. Methods Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. Results Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. Conclusions This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0481-4) contains supplementary material, which is available to authorized users.

Triploidy is a relatively common cause of miscarriage; however, recurrent triploidy has rarely been reported. A healthy 34-year-old woman was ascertained because of 18 consecutive miscarriages with triploidy found in all 5 karyotyped losses. Molecular results in a sixth loss were also consistent with triploidy. Genotyping of markers near the centromere on multiple chromosomes suggested that all six triploid conceptuses occurred as a result of failure to complete meiosis II (MII). The proband's mother had also experienced recurrent miscarriage, with a total of 18 miscarriages. Based on the hypothesis that an inherited autosomal-dominant maternal predisposition would explain the phenotype, whole-exome sequencing of the proband and her parents was undertaken to identify potential candidate variants. After filtering for quality and rarity, potentially damaging variants shared between the proband and her mother were identified in 47 genes. Variants in genes coding for proteins implicated in oocyte maturation, oocyte activation or polar body extrusion were then prioritized. Eight of the most promising candidate variants were confirmed by Sanger sequencing. These included a novel change in the PLCD4 gene, and a rare variant in the OSBPL5 gene, which have been implicated in oocyte activation upon fertilization and completion of MII. Several variants in genes coding proteins playing a role in oocyte maturation and early embryonic development were also identified. The genes identified may be candidates for the study in other women experiencing recurrent triploidy or recurrent IVF failure.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CRC predict the ineffectiveness of EGF receptor-targeted therapy. Previous transcriptional microarray analysis suggests the association between phospholipase Cδ1 (PLCδ1) expression and KRAS mutation status in CRC. However, both the roles and the regulatory mechanisms of PLCδ1 in CRC are not known. Here, we found that the expression of PLCδ1, one of the most basal PLCs, is down-regulated in CRC specimens compared with normal colon epithelium by immunohistochemistry. Furthermore, we examined the roles of PLCδ1 in CRC cell lines that harbor an activating KRAS mutation. Ectopic expression of PLCδ1 in CRC cells induced the expression of E-cadherin, whereas knockdown of PLCδ1 repressed the expression of E-cadherin. Moreover, the overexpression of PLCδ1 suppressed the expression of several mesenchymal genes and reduced cell motility, invasiveness, and in vivo tumorigenicity of SW620 CRC cells. We also showed that PLCδ1 expression is repressed by the KRAS/mitogen-activated protein kinase kinase (MEK) pathway. Furthermore, PLCδ1 suppressed the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 through E-cadherin induction in CRC cells, suggesting the presence of a negative regulatory loop between KRAS/MEK/ERK signaling and PLCδ1. These data indicate that PLCδ1 has tumor-suppressive functions in CRC through E-cadherin induction and KRAS/MEK/ERK signal attenuation.

Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

Phospholipase Cδ4 (PLCδ4) plays a significant role in cell proliferation, tumorigenesis, and in an early stage of fertilization. Despite the characterization of the mammalian PLCδ4, extensive study in aquatic organisms has not been carried out so far. Here, we performed the molecular and biochemical characterization of flatfish Paralichthys olivaceus PLCδ4 ( PoPLCδ4 ) to understand its enzymatic properties and physiological functions. The olive flounder PLCδ4 cDNA has an open reading frame (ORF) of 2,268 bp, and encodes a 755 amino acid polypeptide with a predicted molecular weight of 86 kDa. All the characteristic domains found in mammalian PLCδ isoforms (PH domain, EF hands, an X–Y catalytic region, and a C2 domain) were found to be present in PoPLCδ4 . The mRNA expression analysis of PoPLCδ4 showed that PoPLCδ4 is predominantly expressed in the brain, eye and heart tissues. Like other mammalian PLCδ proteins, the enzyme activity of recombinant PoPLCδ4 to phosphatidylinositol-4,5-bis-phosphate (PIP 2 ) was noted to be concentration- and Ca 2 + -dependent. The structural features and biochemical characteristics of PoPLCδ4 were found to be similar to those of mammalian PLCδ4. This is the first demonstration of the expression analysis and enzymatic characterization of piscine PLCδ4.

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover, and in the regulation of various cellular events. Among the 13 PLC isozymes, PLCδ1 and PLCδ3 share a high sequence homology, and similar tissue distribution. Recent studies with genetically manipulated mice have clarified the importance of these PLC isozymes in a number of tissues. PLCδ1 is required for maintenance of homeostasis in skin and metabolic tissues, while PLCδ3 regulates microvilli formation in enterocytes and the radial migration of neurons in the cerebral cortex of the developing brain. Furthermore, simultaneous loss of PLCδ1 and PLCδ3 in mice causes placental vascular defects, leading to embryonic lethality. Taken together, PLCδ1 and PLCδ3 have unique and redundant roles in various tissues.

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca2+-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking. Background: Annexin A2 is a phospholipid binding protein implicated in endo- and exocytosis. Results: Annexin A2 induces microdomain formation and inward vesicle budding in giant unilamellar vesicles. Conclusion: Specific lipid clustering and vesicle budding is triggered by annexin A2. Significance: Membrane binding by the cytosolic protein annexin A2 can induce the formation of microdomains rich in cholesterol and phosphatidylinositol 4,5-bisphosphate.

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.

Hereditary leukonychia or porcelain nails is a nail dystrophy characterized by whitening of the nail plates in all nails of the hands and feet. It may exhibit an autosomal recessive or autosomal dominant pattern of inheritance. Mutations in the gene PLCD1 have been reported to underlie hereditary leukonychia. In the present study, two Pakistani families with autosomal recessive hereditary leukonychia were investigated. Affected members of the families exhibited characteristic features of hereditary leukonychia with involvement of nails on both the hands and feet. Genotyping using microsatellite markers linked the families to the gene PLCD1 on chromosome 3p22.2. Sequence analysis of the gene detected one novel (p.Ser740ArgfsX19) and one previously reported mutation (p.Arg437X). This study expands spectrum of the mutations in the gene PLCD1 causing hereditary leukonychia.

Phospholipase Cδ3 (PLCδ3) is a key enzyme in phosphoinositide metabolism, however, its physiological function remains unknown. Here we identified the Myosin VI (Myo6) as a binding partner of the PLCδ3. A tail region containing IQ motif and the cargo-binding domain of Myo6, and the C2 domain and PH domain of PLCδ3 were responsible sites for the interaction. Since Myo6 has been well analyzed as one of the "deafness genes" in mouse and human, we examined the expression pattern of PLCδ3 mRNA in the inner ear. In situ hybridization analysis indicated that both Myo6 and PLCδ3 were clearly and limitedly co-expressed in the inner and outer hair cells in the cochlea. Although actin structure of the stereocilia of hair cells seemed to be normal and no detectable hearing defect was observed in PLCδ3 knockout (KO) mice, stable PLCδ3 knockdown in Caco-2 colonic carcinoma cells caused abnormal actin structure of microvilli. In addition, dramatic decrease in expression of Myo6 was observed in intestine of PLCδ3KO mice, where microvilli structure is well developed. These results indicate that PLCδ3 could participate in stability of microvilli structure via regulating and anchoring of Myo6 to plasma membrane.

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.

Located at the important tumor suppressor locus, 3p22, PLCD1 encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movements. We identified PLCD1 as a downregulated gene in aerodigestive carcinomas through expression profiling and epigenetic characterization. We found that PLCD1 was expressed in all normal adult tissues but low or silenced in 84% (16/19) gastric cancer cell lines, well correlated with its CpG island (CGI) methylation status. Methylation was further detected in 62% (61/98) gastric primary tumors, but none of normal gastric mucosa tissues. PLCD1 methylation was significantly correlated with tumor high stage. Detailed methylation analysis of 37 CpG sites at the PLCD1 CGI by bisulfite genomic sequencing confirmed its methylation. PLCD1 silencing could be reversed by pharmacological demethylation with 5-aza-2'-deoxycytidine, indicating a direct epigenetic silencing. Ectopic expression of PLCD1 in silenced gastric tumor cells dramatically inhibited their clonogenicity and migration, possibly through downregulating MMP7 expression and hampering the reorganization of cytoskeleton through cofilin inactivation by phosphorylation. Thus, epigenetic inactivation of PLCD1 is common and tumor-specific in gastric cancer, and PLCD1 acts as a functional tumor suppressor involved in gastric carcinogenesis.

that differ in theirtissue expression patterns and mechanisms of regulation (Fig. 1), not including numerous splicedvariants (Suh et al., 2008). A common catalytic core comprises a TIM barrel structure that is almostalways linked directly to a C2 domain. Additional domains, specific to each group have been identifiedthat confer a range of regulatory interactions with phosphoinositides, small or large GTP-binding (G)proteins, protein kinases, or other proteins that either modulate, or are modulated by, PLC. For thepurposes of this discussion, the most notable are the PH domains of PLC

The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1 ( -/- ) knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+- and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely. Although phosphoinositides regulate many cell functions unrelated to Ca2+ signaling there are still many open questions even in the Ca2+ field that would benefit from single cell monitoring of PtdIns(4,5)P2 or InsP3 changes during agonist stimulation. This chapter is designed to provide practical guidance as well as some theoretical background on measurements of phosphoinositides in live cells using protein domain-GFP chimeras that could be also useful for people working on calcium signaling.

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.

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2levels in32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

Phospholipase C (PLC) and the small G protein RhoA are vital elements for the contraction of vascular smooth muscle cells. The available evidence points to altered PLC-delta1 activity as an element determining enhanced vascular tone in hypertension; however, the factor(s) responsible for increased PLC activity remains unknown. There is the data indicating that RhoA inhibits PLC-delta1 and factors downmodulating RhoA activate phospholipase. In the present study, we explore an impact of a newly identified human ARHGAP6 protein possessing GTPase stimulating activity for RhoA on the catalytic properties of PLC-delta1. Under in vitro conditions, ARHGAP6 protein activated PLC-delta1. ARHGAP6 protein bound PLC-delta1 and regulated its activity by masking the binding sites for inhibitory phospholipids. Moreover, ARHGAP6 increased the V(max) of PLC-delta1 and enhanced its response to Ca(2+) stimulation. A Western blot of immunoprecipitates from Cos-7 cells transfected with pcDNA3-ARHGAP6 and pcDNA3-PLCdelta1 showed the presence of ARHGAP6/PLC-delta1 complexes. The activity of PLC in cells overexpressing ARHGAP6 increased approximately 6-fold compared to control cells. The examination of ARHGAP6 expression in mononuclear cells isolated from the blood of patients with hypertension showed increased ARHGAP6 mRNA and protein levels compared to age-matched normotensive subjects. Enhanced expression of ARHGAP6 was associated with an elevated level of PLC activity and increased levels of IP(3) (1.6-fold) and DAG (2.3-fold). In summary, our data indicate that ARHGAP6 protein binds to and up regulates PLC-delta1 both under in vitro and in vivo conditions. Moreover, the elevated expression of ARHGAP6 provides possible explanation for the altered activity of PLC-delta1 in hypertension.

The delta4 isoform of phospholipase C (PLC-delta4) is thought to be associated with various cellular functions and disease status. However, little is known about how its function is controlled in cells, particularly in terms of the regulation of its expression. To understand the regulation mechanisms of the PLC-delta4 gene transcription, the 5'-flanking region (-2046 approximately +5) (the nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank/DDBJ data bank under accession numbers DQ302751) of the human PLC-delta4 gene was isolated from human genomic DNA. It was a TATA-less promoter with very GC-rich sequences near the transcription start site. The activity of the PLC-delta4 promoter was shown in various human and mouse cell lines by luciferase reporter assay. Serial deletion analysis identified the core promoter region as being between -402 and -67, in which an E-box and an AP-1 binding site played important roles in the promoter activity. In addition, we also showed that 12-O-tetradecanoylphorbol-1,3-acetate (TPA), a PKC activator and tumor promoter, induced the activity of the PLC-delta4 promoter via the AP-1 binding site. In summary, this study identified a core promoter region of the hPLC-delta4 gene and the factor binding sites responsible for the promoter activity. These results will provide important new information to further understand the regulatory mechanism of the PLC-delta4 function.

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)-1β. 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-1β 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-1β and Fcγ receptor-mediated phagocytosis in macrophages.

Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

The levels of expression of G-protein alpha(q/11) (Galpha(q/11)) subunits and PLC-beta(1-4), -gamma, and -delta(1) isoforms were quantified by Western blot analysis in order to establish their contribution to the patterns of PLC functioning reported here. Quantitative measurements of the levels of Galpha(q/11) subunits in each region were obtained by comparison with known amounts of Escherichia coli expressed recombinant Galpha(q) subunits. Quantitative analysis indicated that Galpha(q/11) subunits are abundant polypeptides in human brain, with values ranging from about 1200 ng/mg in cerebral cortex to close to 900 ng/mg of membrane protein in caudate. In cerebral cortical membranes, the PLC-beta(1) isoform was more abundant than in caudate membranes. The highest levels of PLC-beta(2) expression were detected in caudate membranes. PLC-beta(3) was little expressed, and there were no significant differences in the relative values between both brain regions. Finally, the levels of the PLC-beta(4) isoform were significantly lower in caudate than in cortical membranes. It is concluded that although most of these data represent relative, not absolute, measures of protein levels within these regions, they contribute nonetheless to the significant differences observed in signaling capacities through the PLC system in both human brain regions.

Phosphoinositides (PIs) and proteins involved in the PI signaling pathway are distributed in the nucleus as well as at the plasma membrane and in the cytoplasm, although their nuclear localization mechanisms have not been clarified in detail. Generally, proteins that shuttle between the cytoplasm and nucleus contain nuclear localization signal (NLS) and nuclear export signal (NES) sequences for nuclear import and export, respectively. They bind to specific carrier proteins of the importin/exportin family and are transported to and from the nucleus. Thus there is a steady state shuttling of the cargo molecules to and from the nucleus, and the shift in equilibrium determines their nuclear or cytoplasmic localization. Our previous studies have shown that phospholipase C (PLC)-δ1, regarded as having cytoplasmic- or plasma membrane-bound localization, accumulates in the nucleus when its NES sequence is disrupted. In addition, a cluster of positively charged residues on the surface of the catalytic barrel is important for nuclear import. In quiescent cells, the shuttling equilibrium seems to be shifted to the nuclear export of PLCδ1. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling of PLCδ1 will be discussed. It is important to know when and how they are regulated. A shift in the equilibrium in a certain stage of the cell cycle or by external stimuli is possible and resulting changes in the intra-nuclear environments (or architectures) may alter proliferation and differentiation patterns. Evidences support the idea that an increase in the levels of intracellular Ca2+ shifts the equilibrium to the nuclear import of PLCδ1. A myriad of external stimuli have also been reported to change the nuclear PI metabolism following accelerated accumulation in the nucleus of other phospholipases such as phospholipase A2 and phospholipase D in addition to PLC isoforms such as PLCβ1 and PLCγ1. The consequence of the nuclear accumulation of PLC is also discussed. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.

Aim: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase Cdelta 1 (PLCdelta 1 PH) on inhibition of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] by neomycin. Methods: A fusion construct of green fluorescent protein (GFP) and PLCdelta 1 PH (PLCdelta 1 PH-GFP), which is known to bind PtdIns(4,5)P 2 specifically, together with laser-scanning confocal microscopy, was used to trace PtdIns(4,5)P 2 translocation. Results: Stimulation of the type 1 muscarinic receptor and the bradykinin 2 receptor induced a reversible PLCdelta 1 PH-GFP translocation from the membrane to the cytosol in COS-7 cells. PLC inhibitor U73122 blocked the translocation. Wortmannin, a known PtdIns kinase inhibitor, did not affect the translocation induced by ACh, but blocked recovery after translocation, indicating that PtdIns(4,5)P 2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLCdelta1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P 2 hydrolysis. However, in the absence of PLCdelta 1 PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P 2 by PLC. Conclusion: Although PLCdelta 1 PH and neomycin bind to PtdIns(4,5)P 2 in a similar way, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P 2 hydrolysis.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLCdelta1 knockout mice and found that PLCdelta1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLCdelta1 knockout mice, even though PLCdelta1 is broadly distributed. Here, we report that mice lacking both PLCdelta1 and PLCdelta3 died at embryonic day 11.5 (E11.5) to E13.5. PLCdelta1/PLCdelta3 double-knockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLCdelta1/PLCdelta3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLCdelta1 and PLCdelta3 are essential in trophoblasts for placental development.

Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.

Second messengers generated from membrane lipids play a critical role in signaling and control diverse cellular processes. Despite being one of the most evolutionarily conserved of all the phosphoinositide-specific phospholipase C (PLC) isoforms, a family of enzymes responsible for hydrolysis of the membrane lipid phosphatidylinositol bisphosphate, the mechanism of PLC-delta1 activation is still poorly understood. Here we report a novel regulatory mechanism for PLC-delta1 activation that involves direct interaction of the small GTPase Ral and the universal calcium-signaling molecule calmodulin (CaM) with PLC-delta1. In addition, we have identified a novel IQ type CaM binding motif within the catalytic region of PLC-delta1 that is not found in other PLC isoforms. Binding of CaM at the IQ motif inhibits PLC-delta1 activity, while addition of Ral reverses the inhibition. The overexpression of various Ral mutants in cells potentiates PLC-delta1 activity. Thus, the Ral-CaM complex defines a multifaceted regulatory mechanism for PLC-delta1 activation.

Galpha(h), also known as transglutaminase II, has GTPase as well as transglutaminase activities. To better understand the factors affecting these dual enzymatic activities, we examined the optimal pH (at 25 degrees C) and thermal stability (at 37 degrees C) of the activities using membranous Galpha(h) from mouse heart. The optimum pH for the GTPase activity of Galpha(h) is approximately 7.0. As well, the GTP binding activity of Galpha(h) is more thermostable at pH 7.0 than that at pH 9.0. Consistent with these observations on the GTPase function of Galpha(h), both the phospholipase C-delta1 activity and the yield of co-immunoprecipitation of Galpha(h)-coupled phospholipase C-delta1 in alpha(1)-adrenoceptor/Galpha(h)/phospholipase C-delta1 complex preparations were enhanced by incubation with an alpha(1)-agonist, phenylephrine, at pH 7.0. On the other hand, the transglutaminase activity of Galpha(h) is higher in the basic pH range with an optimum activity at pH approximately 9.0. Also, the transglutaminase activity of Galpha(h) is more thermostable at pH 9.0 than that at pH 7.0. These results indicate not only pH as a modulator for the dual functions of Galpha(h), but also provide direct evidence for the involvement of pH in the Galpha(h)-mediated alpha(1)-adrenoceptor signaling system in vitro.

Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to its actin-based motility. In Listeria-infected PtK2 cells Akt-pleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C delta (PLC delta)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P3 by expression of Akt-PH-GFP and available PtdIns(4,5)P2 by expression of PLC delta-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC delta-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P3 production, indicating a central role for PI 3-kinases in Listeria intracellular actin-based motility and filopod formation.

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCdelta, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCdelta was isolated by screening the cDNA library of mud loach liver and using the 5'-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCdelta gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCdelta isozymes (PH domain, EF-hands, X-Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCdelta shares relatively high sequence identity with mammalian PLCdelta1 (51-52%) and catfish PLCdelta (64%). The recombinant ML-PLCdelta protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni(2+)-NTA affinity chromatography. The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP(2)) and its activity was Ca(2+)-dependent, which was similar to mammalian PLCdelta isozymes.

A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cdelta1 (PLCdelta1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCdelta1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 μM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.

Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, including fertilization and development of the embryo. One of the key mechanisms involved in triggering intracellular calcium release is the generation of the second messenger inositol-1,4,5-phosphate (IP(3)) by the phospholipase C (PLC) class of enzymes. Although five distinct forms of PLC have been identified in mammals (beta, gamma, delta, epsilon, and zeta), only one, PLCgamma, has thus far been detected in echinoderms. In the present study, we describe the isolation of a cDNA encoding a novel PLC isoform of the delta (delta) subclass, PLC-deltasu, from the egg of the Pacific purple sea urchin Strongylocentrotus purpuratus. We also demonstrate the presence of this PLC within the sperm and in the early embryo. The PLC-deltasu cDNA (2.44kb) encodes a 742 amino acid polypeptide with an open reading frame of 84.6kDa and a pI of 6.04. All of the characteristic domains found in mammalian PLCdelta isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are present in PLC-deltasu. A homology search revealed that PLC-deltasu shares most sequence identity with bovine PLCdelta2 (39%). We present evidence that PLC-deltasu is expressed in unfertilized eggs, fertilized eggs, and in the early embryo. In addition to Northern and polymerase chain reaction (PCR) analyses, in situ hybridization experiments further demonstrated that the embryonic regions within which the PLC-deltasu transcript can be detected during early embryonic development are associated with the highest levels of proliferative activity, suggesting a possible involvement with metabolism or cell cycle regulation.

Axonal transport of membranous organelles such as mitochondria is essential for neuron viability and function. How signaling mechanisms regulate or influence mitochondrial distribution and transport is still largely unknown. We observed an increase in the distal distribution of mitochondria in neurons upon the expression of pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ-PH) and spectrin (spectrin-PH). Quantitative analysis of mitochondrial transport showed that specific binding of PH domains to phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) but not 3′ phosphorylated phosphatidylinositol species enhanced plus-end–directed transport of mitochondria two- to threefold and at the same time decreased minus-end–directed transport of mitochondria along axonal microtubules (MTs) without altering the overall level of motility. Further, the velocity and duration of mitochondrial transport plus the association of molecular motors with mitochondria remained unchanged by the expression of PH domains. Thus, PtdIns(4,5)P2-specific PH domains caused an increase in distal mitochondria by disturbing the balance of plus- and minus-end–directed transport rather than directly affecting the molecular machinery involved. Taken together our data reveal that level and directionality of transport are separable and that PtdIns(4,5)P2 has a novel role in regulation of the directionality of axonal transport of mitochondria.