PLCD3 promotes malignant cell behaviors in esophageal squamous cell carcinoma via the PI3K/AKT/P21 signaling.

BACKGROUND: Phospholipase C Delta 3 (PLCD3) is a member of phospholipase C(PLC) Protein and PLCD3 protein plays a prominent role in many cancers. However, little is known about the role of PLCD3 in esophageal squamous cell carcinoma (ESCC). MATERIAL AND METHODS: We analyzed PLCD3 mRNA and protein expression in ESCC tissues and cell lines by immunohistochemistry, quantitative real-time PCR, and western blot. The correlation between PLCD3 expression and clinicopathological characteristics was also analyzed. CCK8, colony formation, wound-healing, and transwell assays were conducted to measure cell functional alternations. Flow cytometry was performed to assess the apoptosis rate and cell cycle caused by PLCD3 knockdown. Xenograft models in nude mice to clarify the role of PLCD3 in ESCC. Key proteins in the PI3K / AKT signaling pathway after treatment of ECA109 and KYSE150 cells with the AKT inhibitor MK2206 were analyzed by western blot. RESULTS: PLCD3 was highly expressed in ESCC tissues and cell lines. PLCD3 expression levels correlated with pathologic stage and lymphatic metastasis. PLCD3 knockdown inhibited cell proliferation, migration, invasion, promoted apoptosis, and caused the cell cycle arrest in the G1 phase. PLCD3 overexpression promoted cell proliferation, migration, and invasion. In vivo experiments with xenografts demonstrated that PLCD3 promoted ESCC tumorigenesis. Finally, Overexpression of PLCD3 activated the PI3K / AKT / P21 signaling. CONCLUSION: PLCD3 promotes malignant cell behaviors in esophageal squamous cell carcinoma via the PI3K/AKT/P21 signaling and could serve as a potential target for ESCC treatment.

MicroRNA-191 regulates oral squamous cell carcinoma cells growth by targeting PLCD1 via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Studies have shown that microRNA-191 (miR-191) is involved in the development and progression of a variety of tumors. However, the function and mechanism of miR-191 in oral squamous cell carcinoma (OSCC) have not been clarified. METHODS: The expression level of miR-191 in tumor tissues of patients with primary OSCC and OSCC cell lines were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. OSCC cells were treated with miR-191 enhancers and inhibitors to investigate the effects of elevated or decreased miR-191 expression on OSCC cells proliferation, migration, cell cycle, and tumorigenesis. The target gene of miR-191 in OSCC cells were analyzed by dual-Luciferase assay, and the downstream signaling pathway of the target genes was detected using western blot assay. RESULTS: The expression of miR-191 was significantly upregulated in OSCC tissues and cell lines. Upregulation of miR-191 promoted proliferation, migration, invasion, and cell cycle progression of OSCC cells, as well as tumor growth in nude mice. Meanwhile, reduced expression of miR-191 inhibited these processes. Phospholipase C delta1 (PLCD1) expression was significantly downregulated, and negatively correlated with the expression of miR-191 in OSCC tissues. Dual-Luciferase assays showed that miR-191-5p could bind to PLCD1 mRNA and regulate PLCD1 protein expression. Western blot assay showed that the miR-191 regulated the expression of ß-catenin and its downstream gene through targeting PLCD1. CONCLUSION: MicroRNA-191 regulates oral squamous cell carcinoma cells growth by targeting PLCD1 via the Wnt/ß-catenin signaling pathway. Thus, miR-191 may serve as a potential target for the treatment of OSCC.

Circ_0003747 promotes thyroid cancer progression by sponging miR-338-3p to upregulate PLCD3 expression.

The circular RNAs (circRNAs) involved in competitive endogenous RNA (ceRNA) mechanism are critical modulators affecting pathogenesis of thyroid carcinoma (TC). The study's goal was to investigate the effects of circ 0003747 on the biological progression of papillary thyroid cancer (PTC). Normal thyroid cells Nthy-ori3-1 and TC derived cell lines were used in our study. Sanger sequencing and RNase R treatment were utilized for validating the circular structure of circ_0003747. In our work, circ_0003747 was found to be highly expressed in TC cells. Circ_0003747 knockdown reduced TC cell viability, proliferation, migration, and invasion while increasing cell apoptosis. Circ_0003747 targeted and negatively regulated miR-338-3p expression. Besides, miR-338-3p interacted with PLCD3 to repress its expression. Overexpression of miR-338-3p inhibited TC cell progression, and PLCD3 reversed these effects. Furthermore, PLCD3 overexpression reversed the effects of circ_0003747 knockdown on TC cells. Additionally, the knockdown of circ_0003747 remarkably suppressed tumour size and growth, restrained PLCD3 expression and promoted miR-338-3p expression in nude mice. In conclusion, circ_0003747 facilitated the biological progression of TC by modulating the miR-338-3p/PLCD3 axis, and it may be a new target for TC treatment. [Figure: see text]Abbreviations: TC: Thyroid carcinoma; PTC: Papillary thyroid carcinoma; CircRNAs: Circular RNAs; MiRNA: MicroRNA; EMT: Epithelial-mesenchymal transition; HCC: Hepatocellular carcinoma; PLCD3: Phospholipase C Delta 3; CeRNA: Competitive endogenous RNA.

lncRNA MIR503HG Targets miR-191-5p/PLCD1 Axis and Negatively Modulates Apoptosis, Extracellular Matrix Disruption, and Inflammation in Abdominal Aortic Aneurysm.

As the most prevalent subtype of aortic aneurysm, abdominal aortic aneurysm (AAA) features the apoptosis, extracellular matrix (ECM) disruption, and inflammation response of vascular smooth muscle cells (VSMCs). Noncoding RNAs (ncRNAs) are crucial factors in AAA progression, while the investigations have not been fully explained. miR-191-5p upregulation is found in aortic aneurysm. However, its role in AAA has not been addressed. This research purposed to excavate the possible and associated molecular axis of miR-191-5p in AAA. In our study, miR-191-5p level was detected to be high in the tissues from AAA patients in comparison with the control group. After miR-191-5p expression was enhanced, cell viability was repressed, cell apoptosis was boosted, and ECM disruption and the inflammation response were fortified. Furthermore, the relationship among MIR503HG, miR-191-5p, and phospholipase C delta 1 (PLCD1) in VSMCs was disclosed via mechanism assays. Decreased MIR503HG lacked the inhibition on miR-191-5p targeting PLCD1, resulting in downregulation of PLCD1, which facilitated the progression of AAA. Thus, targeting MIR503HG/miR-191-5p/PLCD1 pathway will provide an additional method for the cure of AAA patients.

MiR-17-3p Facilitates Aggressive Cell Phenotypes in Colon Cancer by Targeting PLCD1 Through Affecting KIF14.

Colon cancer (CC) is a common and lethal cancer to be further elucidated. Accumulating studies elaborated the crucial role of miRNAs differentially expressed in cancer cell growth. In the present study, differentially expressed miRNAs related to CC were screened by the bioinformatics methods on the strength of TCGA database. Highly expressed miR-17-3p was proved to notably influence CC cell proliferative, migratory, invasion, and apoptotic levels. By using TargetScan and miRTarBase databases, phospholipase C delta 1 (PLCD1) was predicted as a target downstream of miR-17-3p, and their binding site was predicted. Through TCGA database, low expression of PLCD1 and its significant negative correlation with miR-17-3p were identified in CC. Dual-luciferase reporter gene analysis ascertained the targeting relationship between miR-17-3p and PLCD1. Cell Counting Kit-8, colony formation, and transwell assays were introduced to detect CC cell malignant progression. Flow cytometry was applied to detect CC cell apoptosis. As result revealed, miR-17-3p was markedly highly expressed, and PLCD1, the target of miR-17-3p, was remarkably lowly expressed in CC cells. Forced expression of miR-17-3p facilitated CC cell proliferation, migration, invasion, and suppressed apoptosis. Biological roles of upregulating miR-17-3p in the colon cancer cells were markedly weakened by over-expressing PLCD1 simultaneously. MiR-17-3p regulated CC cell malignant progression, as well as apoptosis by targeting PLCD1. Moreover, KIF14 was extensively considered as an involved tumor-promoting gene that could be affected by miR-17-3p/PLCD1 axis based on BioGRID analysis and CO-IP assay. Concludingly, this study exhibited that miR-17-3p facilitated CC progression by PLCD1 downregulation.

Circular RNA circ-PLCD1 functions as a tumor suppressor in non-small cell lung cancer by inactivation of PI3K/AKT signaling pathway.

Circular RNAs (circRNAs) are emerging as crucial regulators in tumorigenesis and aggressive progression. However, their biological roles in non-small cell lung cancer (NSCLC) remain largely unknown. Here, by performing circRNA high throughput sequencing in 4 paired NSCLC and normal tissues, we found a NSCLC-associated circRNA, circ-PLCD1, which was evidently downregulated in NSCLC tissues and cell lines. Circ-PLCD1 was transcriptionally activated by tumor-inhibiting protein p53, and exogenous expression of circ-PLCD1 inhibited NSCLC cell proliferation, invasion and induced apoptosis. Mechanistically, circ-PLCD1 acted as a competitive endogenous RNA (ceRNA) to sponge miR-375 and miR-1179 and elevate PTEN, a well-known inhibitor of oncogenic PI3K/AKT signaling, thereby repressing NSCLC tumorigenesis. Importantly, we also identified this ceRNA regulatory axis of circ-PLCD1/miR-375/miR-1179/PTEN in vivo by establishing a xenograft tumor model. Clinically, NSCLC patients with low circ-PLCD1 expression had larger tumor size, later clinical stage and shorter survival time than those with high circ-PLCD1 expression. Altogether, our findings reveal the important tumor suppressive role of circ-PLCD1 in NSCLC, reactivation of this circRNA may be considered as a novel therapeutic avenue for patient with NSCLC.

The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1.

BACKGROUND: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. METHODS: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. RESULTS: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. CONCLUSION: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

Optical Control of Phosphoinositide Binding: Rapid Activation of Subcellular Protein Translocation and Cell Signaling.

Cells utilize protein translocation to specific compartments for spatial and temporal regulation of protein activity, in particular in the context of signaling processes. Protein recognition and binding to various subcellular membranes is mediated by a network of phosphatidylinositol phosphate (PIP) species bearing one or multiple phosphate moieties on the polar inositol head. Here, we report a new, highly efficient method for optical control of protein localization through the site-specific incorporation of a photocaged amino acid for steric and electrostatic disruption of inositol phosphate recognition and binding. We demonstrate general applicability of the approach by photocaging two unrelated proteins, sorting nexin 3 (SNX3) and the pleckstrin homology (PH) domain of phospholipase C delta 1 (PLCδ1), with two distinct PIP binding domains and distinct subcellular localizations. We have established the applicability of this methodology through its application to Son of Sevenless 2 (SOS2), a signaling protein involved in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Upon fusing the photocaged plasma membrane-targeted construct PH-enhanced green fluorescent protein (EGFP), to the catalytic domain of SOS2, we demonstrated light-induced membrane localization of the construct resulting in fast and extensive activation of the ERK signaling pathway in NIH 3T3 cells. This approach can be readily extended to other proteins, with minimal protein engineering, and provides a method for acute optical control of protein translocation with rapid and complete activation.

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

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

Phospholipase C Delta 3 inhibits apoptosis and promotes proliferation, migration, and invasion of thyroid cancer cells via Hippo pathway.

In recent decades, the incidence of thyroid cancer (TC) has rapidly increased, leading us to explore the complex underlying mechanisms. We identified the gene Phospholipase C Delta 3 (PLCD3) as a potential oncogene in TC by conducting the whole transcriptome sequencing. Our study is to understand the oncogenic role of PLCD3 in TC. We verified the overexpression of PLCD3 in TC from The Cancer Genome Atlas, Gene Expression Omnibus databases, and a locally validated cohort. Clinical correlation analysis showed that PLCD3 expression was related to histological type, T stage, lymph node metastasis (LNM), and disease stage. The high expression of PLCD3 could be a distinguishing factor for TC and its LNM. The biological function was examined using small interfering RNA-transfected TC cell lines. Silenced PLCD3 could inhibit colony formation, migration, and invasion ability and promote apoptosis of TC cell lines. PLCD3 silencing reversed the epithelial-mesenchymal transition but induced the apoptotic progress. Further exploration revealed that PLCD3 might be associated with critical genes of the Hippo pathway. The expressions of RHOA, YAP1/TAZ, and their downstream targets were decreased significantly when PLCD3 was down-regulated. YAP1 overexpression rescued the tumor-suppressive effect caused by PLCD3 silencing. This study demonstrates that PLCD3 is an oncogene that supports tumorigenesis and progression in TC, and PLCD3 may be a potential target gene for TC treatment.

The Gαh/phospholipase C-δ1 interaction promotes autophagosome degradation by activating the Akt/mTORC1 pathway in metastatic triple-negative breast cancer.

Lung metastasis (LM) is commonly found in triple-negative breast cancer (TNBC); however, the molecular mechanism underlying TNBC metastasis to lungs remains largely unknown. We thus aimed to uncover a possible mechanism for the LM of TNBC. Here we show that the phosphorylation of Akt and mTORC1 was positively but the autophagy activity was negatively correlated with endogenous Gαh levels and cell invasion ability in TNBC cell lines. Whereas the knockdown of Gαh, as well as blocking its binding with PLC-δ1 by a synthetic peptide inhibitor, in the highly invasive MDA-MB231 cells dramatically suppressed Akt/mTORC1 phosphorylation and blocked autophagosome degradation, the overexpression of Gαh in the poorly invasive HCC1806 cells enhanced Akt/mTORC1 phosphorylation but promoted autophagosome degradation. The pharmaceutical inhibition of autophagy initiation by 3-methyladenine was found to rescue the cell invasion ability and LM potential of Gαh-silenced MDA-MB231 cells. In contrast, the inhibition of mTORC1 activity by rapamycin suppressed autophagosome degradation but mitigated the cell invasion ability and LM potential of Gαh-overexpressing HCC1806 cells. These findings demonstrate that the induction of autophagy activity or the inhibition of Akt-mTORC1 axis provides a useful strategy to combat the Gαh/PLC-δ1-driven LM of TNBC.

Exogenous NAD+ Stimulates MUC2 Expression in LS 174T Goblet Cells via the PLC-Delta/PTGES/PKC-Delta/ERK/CREB Signaling Pathway.

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)-δ and prostaglandin E synthase (PTGES) expression, which was inhibited by the appropriate inhibitors. Among the protein kinase C (PKC) isozymes, PKC-δ 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-δ/PTGES/PKC-δ/ERK/CREB signaling pathway.

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

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

Stochastic geometry sensing and polarization in a lipid kinase-phosphatase competitive reaction.

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.

The phosphoinositide hydrolase phospholipase C delta1 inhibits epithelial-mesenchymal transition and is silenced in colorectal cancer.

In this study, we found that the phospholipase C delta1 (PLCD1) protein expression is reduced in colorectal tumor tissues compared with paired surgical margin tissues. PLCD1-promoted CpG methylation was detected in 29/64 (45%) primary colorectal tumors, but not in nontumor tissues. The PLCD1 RNA expression was also reduced in three out of six cell lines, due to PLCD1 methylation. The ectopic expression of PLCD1 resulted in inhibited proliferation and attenuated migration of colorectal tumor cells, yet promoted colorectal tumor cell apoptosis in vitro. We also observed that PLCD1 suppressed proliferation and promoted apoptosis in vivo. In addition, PLCD1 induced G1/S phase cell cycle arrest. Furthermore, we found that PLCD1 led to the downregulation of several factors downstream of ß-catenin, including c-Myc and cyclin D1, which are generally known to be promoters of tumorigenesis. This downregulation was caused by an upregulation of E-cadherin in colorectal tumor cells. Our findings provide insights into the role of PLCD1 as a tumor suppressor gene in colorectal cancer (CRC), and demonstrate that it plays significant roles in proliferation, migration, invasion, cell cycle progression, and epithelial-mesenchymal transition. On the basis of these results, tumor-specific methylation of PLCD1 could be used as a novel biomarker for early detection and prognostic prediction in CRC.

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.

A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins.

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.

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

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

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

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.

PLCD3, a flotillin2-interacting protein, is involved in proliferation, migration and invasion of nasopharyngeal carcinoma cells.

Phospholipase C (PLC) is a pivotal enzyme in the phosphoinositide pathway that promotes the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), to participate in eukaryotic signal transduction. Several PLC isozymes are associated with cancer, such as PLC-ß1, PLC-δ1, PLC-ε and PLC-γ1. However, the role of PLC-δ3 (PLCD3) in nasopharyngeal carcinoma (NPC) has not been investigated to date. In our previous study, we demonstrated that flotillin2 (Flot2) plays a pro-neoplastic role in NPC and is involved in tumour progression and metastasis. In the present study, we screened the interacting proteins of Flot2 using the yeast two-hybrid (Y2H) method and verified the interaction between PLCD3 and Flot2 by co-immunoprecipitation. We also investigated the biological functions of PLCD3 in NPC. Inhibition of PLCD3 expression impaired the malignant potential of 5-8F, a highly metastatic NPC cell line, by restraining its growth, proliferation, mobility and migration. The present study demonstrated that PLCD3 may be an oncogenic protein in NPC and that it plays an important role in the progression of NPC partially by interacting with Flot2.