OBJECTIVETo investigate the effect of phosphoinositide 4-phosphate (PI4P) on human glioma U87 cells and the mechanism of action of PI4P in the development of human glioma through the overexpression or silencing of PI4P in human glioma U87 cells, and to provide a new target for basic research and clinical treatment of glioma.

METHODSLV-Helper1, LV-Helper2, pWPXLd-PI4P, and pLL3.7-shPI4P were used to package pWPXLd-PI4P and pLL3.7-shPI4P lentiviruses. The U87-GFP (PI4P-overexpression control group), U87-GFP-PI4P (PI4P-overexpression experimental group), U87-Scramble (PI4P-silencing control group), and U87-shPI4P (PI4P-silencing experimental group) cell lines were established. Wound-healing assay and Transwell assay were used to evaluate cell migration and invasion, and Western blot was used to measure the expression of PI4P in each group.

RESULTSWestern blot detected the expression of exogenous PI4P in the U87-GFP-PI4P cell line, and the U87-shPI4P cell line showed reduced expression of PI4P compared with the U87-Scramble cell line in the control group. The U87-GFP-PI4P cell line with PI4P overexpression had a significantly stronger ability of migration than the U87-GFP cell line in the control group (P<0.01); the U87-shPI4P cell line with PI4P silencing had a reduced ability of migration than the U87-Scramble cell line in the control group (P<0.01). The U87 cell line with PI4P overexpression had a significantly stronger invasion ability than the control group (P<0.05); after PI4P silencing, the experimental group showed a significant reduction in invasion ability compared with the control group (P<0.05).

CONCLUSIONSIn human glioma U87 cells, PI4P can promote the invasion and migration of glioma cells and may become a new target in the basic research and clinical treatment of glioma.

Cell Line, Tumor ; Cell Movement ; drug effects ; Glioma ; pathology ; Humans ; Neoplasm Invasiveness ; Phosphatidylinositol Phosphates ; pharmacology

Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.
Animals ; Fluorescent Antibody Technique ; Mice ; Mutation/genetics ; Neurofilament Proteins/genetics/*metabolism ; Phosphatidylinositol Phosphates/*metabolism ; Phospholipase C gamma/metabolism ; *Protein Multimerization

A novel unsaturated polyphosphoester (UPPE) was devised in our previous research, which is a kind of promising scaffold for improving bone regeneration. However, the polymerization process of UPPE scaffolds was unfavorable, which may adversely affect the bioactivity of osteoinductive molecules added if necessary, such as recombinant human bone morphogenetic protein-2 (rhBMP2). The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2 (UPB) and to investigate the bioactivity of rhBMP2 in this scaffold. Furthermore, the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro. A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres, and then the microspheres were added to UPPE for synthesizing UPB scaffold. The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy. The cumulative release of UPB scaffolds was detected by using ELISA. The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells (bMSCs) seeded on the surface of UPB scaffolds. The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates (ALP) activity in bMSCs seeded. The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2. The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h. bMSCs attached and grew on the surface of soaked UPB scaffolds, exerting well biocompatibility. The ALP activity of bMSCs seeded was significantly enhanced, indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds. It was concluded that UPB scaffolds have low cytotoxicity, good biocompatibility and preserve bioactivity of rhBMP2. UPB scaffolds are promising in improving bone regeneration.
Bone Morphogenetic Protein 2 ; chemistry ; pharmacology ; Bone Regeneration ; drug effects ; Humans ; Lactic Acid ; chemistry ; pharmacology ; Phosphatidylinositol Phosphates ; chemistry ; pharmacology ; Polyglycolic Acid ; chemistry ; pharmacology ; Tissue Scaffolds

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.
1-Phosphatidylinositol 3-Kinase/metabolism ; Alkyl and Aryl Transferases/metabolism ; Animals ; Anticholesteremic Agents/pharmacology ; Cells, Cultured ; Enzyme Inhibitors/metabolism/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression Regulation, Enzymologic/*drug effects ; I-kappa B Kinase/antagonists & inhibitors/metabolism ; Macrophages, Alveolar/cytology/*drug effects/*enzymology ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Polyisoprenyl Phosphates/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Sesquiterpenes/metabolism ; Signal Transduction/physiology ; Simvastatin/*pharmacology ; Smoke/*adverse effects ; *Tobacco/adverse effects/chemistry

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.
Calcium ; Cytoplasm ; Endoplasmic Reticulum ; GTP-Binding Proteins ; HEK293 Cells ; Inositol ; Phosphatidylinositol 4,5-Diphosphate ; Phospholipases ; Phosphotransferases ; Protein Kinase C ; Transient Receptor Potential Channels ; Type C Phospholipases

Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Kalpha, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Kalpha in PC12 rat pheochromocytoma cells by stable expression of PIP5Kalpha microRNA that significantly reduced PIP5Kalpha expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Kalpha-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Kalpha into PIP5Kalpha KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Kalpha KD, but was attenuated by the reintroduction of PIP5Kalpha. Moreover, exogenously applied PIP2 to PIP5Kalpha KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Kalpha acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.
Animals ; Enzyme Activation/drug effects ; Gene Knockdown Techniques ; Mice ; Nerve Growth Factor/*pharmacology ; Neurites/drug effects/*enzymology ; PC12 Cells ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation/drug effects ; Phosphotransferases (Alcohol Group Acceptor)/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence

OBJECTIVETo test whether tanshinone II A (Tan II A), a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin (lipopolysaccharides, LPS)-induced endothelial injury.

METHODSEndothelial cell injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone II A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase (ROCK) pathway-associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray.

RESULTSTan II A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 (ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, or PIP2 in Western blotting), focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan II A, Y27632 and valsartan.

CONCLUSIONTan II A exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.

Apoptosis ; drug effects ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cytoprotection ; drug effects ; Cytoskeleton ; drug effects ; metabolism ; Diterpenes, Abietane ; chemistry ; pharmacology ; Down-Regulation ; drug effects ; genetics ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; pathology ; Humans ; Integrin alphaV ; metabolism ; Lipopolysaccharides ; Myosin Light Chains ; metabolism ; Oligonucleotide Array Sequence Analysis ; Phosphatidylinositol 4,5-Diphosphate ; metabolism ; Protective Agents ; pharmacology ; Signal Transduction ; drug effects ; Up-Regulation ; drug effects ; genetics ; Vinculin ; metabolism ; rho GTP-Binding Proteins ; metabolism ; rho-Associated Kinases ; metabolism