Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.     

Protein kinase C-dependent stimulation of phospholipase D in phospholipase C-treated fibroblasts

Treatment of [¹⁴C]choline- or [¹⁴C]ethanolamine-labeled NIH 3T3 fibroblasts withBacillus cereus phosphatidylcholine-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective¹⁴C-labeled phospholipids. PLD activity was stimulated by 1.5 U/mL of POLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to similar extents. Treatment of¹⁴C]palmitic acid-labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibroblasts with exogenous PLC results in PKC-dependent activation of PLD.     

Sterol and phospholipid acyl chain alterations inSaccharomyces cerevisiae secretion mutants as a function of temperature stress

Analyses of free sterol, steryl ester and fatty acid components from yeast secretion mutants indicated that free and esterified sterol remained relatively constant over a growth range of 24 C to 34 C. The saturated fatty acid components (16∶0 and 18∶0) increased while the unsaturated fatty acids (16∶1 and 18∶1) decreased as the growth temperature increased. In secretory mutants, fatty acid composition changes are more pronounced than in the wild-type strain. A shift toward increased saturated and decreased unsaturated fatty acid was observed when cells were subjected to a 2-hr temperature upshift to 37 C. Steady-state fluorescence anisotropy data indicated that modifications to the lipid component of yeast plasma membrane produced lipid thermotropic transitions that were 3 C to 6 C higher in yeast cells subjected to thermal stress.     

Production and protein kinase C activation of diacylglycerols containing polymethylene-interrupted PUFA

Sciadonic acid (20∶3, Δ-5c,11c,14c) is a polymethylene-interrupted PUFA (PMI-PUFA) that is present in conifer seeds and known to be incorporated into animal cells and to accumulate in membrane PI as a substitute for arachidonate. In this study, we investigated whether PI having sciadonate could serve as source of DAG that could activate protein kinase C (PKC). When Swiss 3T3 cells cultured with sciadonic acid were stimulated with 100 nM of bombesin, 1-stearoyl-2-sciadonoyl-glycerol (G) and 1-stearoyl-2-arachidonoyl-G were produced. The net increments of these two molecular species of DAG reflected the levels of the two molecular species in the PI in the cells. When cells cultured with juniperonic acid (20∶4, Δ-5c,11c,14c,17c) were stimulated 1-stearoyl-2-juniperonoyl-G was produced in proportion to the level of this molecular species in PI in the cells. We also examined PKC activation by synthetic DAG using a partially purified PKC fraction from rat brain and found that both 1-stearoyl-2-sciadonoyl-G and 1-stearoyl-2-juniperonoyl-G could activate PKC comparably to 1-stearoyl-2-arachidonoyl-G. These results indicate that 1-stearoyl-PI having these C₂₀ PMI-PUFA residues can serve as sources of potential signaling molecules.     

Some phorbol esters might partially resemble bryostatin 1 in their actions on LNCaP prostate cancer cells and U937 leukemia cells

Phorbol 12‐myristate 13‐acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl‐indolactam V to 24 % for phorbol 12,13‐dibenzoate. Dose–response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose–response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin‐like behavior may be obtained from other structural templates.     

The Influence of Polyunsaturated Fatty Acids on the Phospholipase D Isoforms Trafficking and Activity in Mast Cells

The impact of polyunsaturated fatty acid (PUFA) supplementation on phospholipase D (PLD) trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.     

Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells

Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFα) in NIH/3T3 cells. We observed that both PMA and TGFα induced ERK1/ERK2 phosphorylation within 5 min of stimulation. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFα did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA-but not TGFα-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKCα and PKCε, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKCδ isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase (ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKCα and ε isoforms.     

Berberine Inhibits Oxygen Consumption Rate Independent of Alteration in Cardiolipin Levels in H9c2 Cells

Small clinical studies have shown that oral treatment with the plant alkaloid berberine (BBR) reduces blood glucose levels similar to that of metformin and have promoted its use as a novel anti‐diabetic therapy. However, in vitro studies have shown that high concentrations of BBR potently inhibit cell proliferation through inhibition of mitochondrial function. Cardiolipin (Ptd₂Gro) is a key phospholipid required for regulating mitochondrial bioenergetic function. We examined if BBR inhibited oxygen consumption rate in H9c2 cardiac myocytes through alteration in Ptd₂Gro metabolism. Treatment of H9c2 cells with BBR resulted in a rapid (within minutes) concentration‐dependent decrease in the oxygen consumption rate (OCR) as determined using a Seahorse XF24 analyzer. Concentrations of BBR as low as 1 µM were effective in inhibiting OCR. In addition, all concentrations of BBR inhibited the fatty acid‐mediated increase in OCR that was observed in untreated cells. Treatment of H9c2 cells with up to 25 µM BBR for 24 h markedly reduced [³H]thymidine incorporation into cells but did not alter the pool size of Ptd₂Gro. In contrast, 12.5 µM BBR increased [1‐¹⁴C]palmitate incorporation into Ptd₂Gro and 12.5 µM and 25 µM BBR reduced [1‐¹⁴C]oleate incorporation into Ptd₂Gro. Protein kinase C delta (PKCδ) activation through its increased membrane association is known to alter Ptd₂Gro distribution within mitochondria. BBR treatment resulted in a decrease in membrane‐associated PKCδ and attenuated the palmitate‐mediated increase in PKCδ membrane‐association. Thus, BBR treatment of H9c2 cardiac myocytes inhibits cellular OCR independent of alteration in Ptd₂Gro levels.     

Inhibition of TRPM7 Channels Reduces Degranulation and Release of Cytokines in Rat Bone Marrow-Derived Mast Cells

Background: mast cells play an important role in airway inflammation in asthma. The transient receptor potential melastatin-like 7 (TRPM7) channel is expressed in primary human lung mast cells and plays a critical role for cell survival. This study aimed to investigate the role of TRPM7 on degranulation and release of cytokines in rat bone marrow-derived mast cells (BMMCs). Methods: the expression levels of TRPM7 were observed by immunocytochemistry and RT-PCR between normal and asthmatic rat BMMCs. TRPM7-specific shRNA and 2-aminoethoxydiphenyl borate (2-APB) and specific shTRPM7 were used to inhibit the function of TRPM7. Degranulation levels were analyzed by beta-hexosaminidase assay. Histamine, TNF-α, IL-6 and IL-13 levels were measured by ELISA. Results: the expression of TRPM7 was significantly higher in asthmatic rat BMMCs than in the normal control group. After application of 2-APB and down-regulation of TRPM7, the beta-hexosaminidase activity and secretion of histamine, IL-6, IL-13 and TNF-α were significantly decreased in the asthmatic group compared to the control group. Conclusion: this study indicates that TRPM7 channels may be involved in the process of degranulation and release of cytokines in rat bone marrow-derived mast cells.     

Calcium-induced membrane microdomains trigger plant phospholipase D activity

Plant alpha-type phospholipase D proteins are calcium-dependent, lipolytic enzymes. The morphology of the aggregates of their phospholipid substrate fundamentally defines the interaction between the enzyme and the surface. Here we demonstrate that the Ca(2+)-induced generation of membrane microdomains dramatically activates alpha-type phospholipase D from white cabbage. 500-fold stimulation was observed upon incorporation of 10 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles in the presence of Ca(2+) ions. Enhanced association of PLDalpha2 with phospholipid surfaces containing anionic components was indicated by lag phase analysis and film balance measurements. Differential scanning calorimetry showed that the POPA-specific activation correlates with the phase behavior of the POPC/POPA vesicles in the presence of Ca(2+) ions. We conclude from the results that the Ca(2+)-induced formation of POPA microdomains is the crucial parameter that facilitates the binding of PLD to the phospholipid surface and suggest that this effect serves as a cellular switch for controlling PLD activity.     

Designing zinc oxide nanostructures (nanoworms,nanoflowers, nanowalls,and nanorods) by pulsed laser ablation technique for gas-sensing application

In this study, pulsed laser ablation technique, also known as pulsed laser deposition (PLD), is used to design and grow zinc oxide (ZnO) nanostructures (nanoworms, nanowalls, and nanorods) by template/seeding approach for gas-sensing applications. Conventionally, ZnO nanostructures used for gas-sensing have been usually prepared via chemical route, where the 3D/2D nanostructures are chemically synthesized and subsequently plated on an appropriate substrate. However, using pulsed laser ablation technique, the ZnO nanostructures are structurally designed and grown directly on a substrate using a two-step temperature-pressure seeding approach. This approach has been optimized to design various ZnO nanostructures by understanding the effect of substrate temperature in the 300-750°C range under O₂ gas pressure from 10-mTorr to 10 Torr. Using a thin ZnO seed layer as template that is deposited first at substrate temperature of ~300°C at background oxygen pressure of 10 mTorr on Si(100), ZnO nanostructures, such as nanoworms, nanowalls, and nanorods (with secondary flower-like growth) were grown at substrate temperatures and oxygen background pressures of (550°C and 2 Torr), (550°C and 0.5 Torr), and (650°C and 2 Torr), respectively. The morphology and the optical properties of ZnO nanostructures were examined by Scanning Electron Microscope (SEM-EDX), X-ray Diffraction (XRD), and photoluminescence (PL). The PLD-grown ZnO nanostructures are single-crystals and are highly oriented in the c-axis. The vapor-solid (VS) model is proposed to be responsible for the growth of ZnO nanostructures by PLD process. Furthermore, the ZnO nanowall structure is a very promising nanostructure due to its very high surface-to-volume ratio. Although ZnO nanowalls have been grown by other methods for sensor application, to this date, only a very few ZnO nanowalls have been grown by PLD for this purpose. In this regard, ZnO nanowall structures are deposited by PLD on an Al₂O₃ test sensor and assessed for their responses to CO and ethanol gases at 50 ppm, where good responses were observed at 350 and 400°C, respectively. The PLD-grown ZnO nanostructures are very excellent materials for potential applications such as in dye-sensitized solar cells, perovskite solar cells and biological and gas sensors.     

Enhancement in Phospholipase D Activity as a New Proposed Molecular Mechanism of Haloperidol-Induced Neurotoxicity

Membrane phospholipase D (PLD) is associated with numerous neuronal functions, such as axonal growth, synaptogenesis, formation of secretory vesicles, neurodegeneration, and apoptosis. PLD acts mainly on phosphatidylcholine, from which phosphatidic acid (PA) and choline are formed. In turn, PA is a key element of the PLD-dependent secondary messenger system. Changes in PLD activity are associated with the mechanism of action of olanzapine, an atypical antipsychotic. The aim of the present study was to assess the effect of short-term administration of the first-generation antipsychotic drugs haloperidol, chlorpromazine, and fluphenazine on membrane PLD activity in the rat brain. Animals were sacrificed for a time equal to the half-life of the antipsychotic drug in the brain, then the membranes in which PLD activity was determined were isolated from the tissue. The results indicate that only haloperidol in a higher dose increases the activity of phospholipase D. Such a mechanism of action of haloperidol has not been described previously. Induction of PLD activity by haloperidol may be related to its mechanism of cytotoxicity. The finding could justify the use of PLD inhibitors as protective drugs against the cytotoxicity of first-generation antipsychotic drugs like haloperidol.     

The Cross Talk between cGMP Signal Pathway and PKC in Pulmonary Endothelial Cell Angiogenesis

Angiogenic proliferation of vascular endothelial cells is believed to play an important role in pulmonary vascular remodeling in pulmonary arterial hypertension. In the present study, we found that c-GMP (cyclic guanosine monophosphate) inhibited the proliferation and tube formation of pulmonary vascular endothelial cells induced by TGF-β1, and that this process was reversed by PKG (protein kinase G) inhibitor and PKC (protein kinase C) inhibitor. In addition, small interfering RNA (siRNA) targeting ERK also reduced cellular proliferation. Furthermore, western blotting showed that cGMP down-regulated the phosphorylation level of ERK1/2, which was reversed not only by PKG inhibitor but also by PKC inhibitor. Silencing different PKC isoforms showed that PKCΔ, PKCγ and PKCα were involved in ERK phosphorylation, suggesting that PKC kinases have a permissive action. Three subtypes, PKCΔ, PKCγ and PKCα are likely to be involved the phosphorylation suppression of ERK included cGMP. Taken together, these data suggest that ERK phosphorylation mediates the proliferation of pulmonary vascular endothelial cells, and PKC kinases have a permissive action in this process.     

Genomic structure,cloning and expression of two phospholipase D isoenzymes from white cabbage

Phospholipase D (PLD) from cabbage is interesting as biocatalyst in phospholipid transformation. To provide the basis for genetic engineering of the enzyme, gene cloning and sequencing were carried out. We have recently identified two isoenzymes, PLD1 and PLD2, on the basis of their cDNAs, here we describe their genomic structure consisting of 3404 and 3614 bp, respectively. Based on their sequence, PLD1 and PLD2 can be assigned to the α‐type of plant PLDs, they contain two HKD motifs and the C2 domain with a phosphatidylinositol 4,5‐bisphosphate (PIP₂) binding motif. Starting from pld1 cDNA, expression studies were carried out. Whereas expression using constructs with StrepTactin or Glutathion S‐transferase tags was not successful, soluble active enzyme was produced from constructs without tag. pld2 was expressed accordingly. Both enzymes were purified by Ca²⁺‐mediated hydrophobic interaction chromatography to high purity. N‐terminal sequencing of PLD1 and PLD2 revealed that the Met‐free N‐termini of both enzymes correspond to sequences derived from the coding region of the pld1 and pld2 genes, respectively. Both recombinant enzymes showed highest hydrolytic activities at pH 5.5 to 5.6, independent of Ca²⁺ concentration (10—100 mM). The optimum Ca²⁺ concentration was 45 mM for PLD1 and PLD2. Both enzymes showed comparable activities in hydrolysis and transphosphatidylation of phospholipids.     

Is It Possible to Find an Antimicrobial Peptide That Passes the Membrane Bilayer with Minimal Force Resistance? An Attempt at a Predictive Approach by Molecular Dynamics Simulation

There is still no answer to the mechanism of penetration of AMP peptides through the membrane bilayer. Several mechanisms for such a process have been proposed. It is necessary to understand whether it is possible, using the molecular dynamics method, to determine the ability of peptides of different compositions and lengths to pass through a membrane bilayer. To explain the passage of a peptide through a membrane bilayer, a method for preparing a membrane phospholipid bilayer was proposed, and 656 steered molecular dynamics calculations were carried out for pulling 7 amyloidogenic peptides with antimicrobial potential, and monopeptides (homo-repeats consisting of 10 residues of the same amino acid: Poly (Ala), Poly (Leu), Poly (Met), Poly (Arg), and Poly (Glu)) with various sequences through the membrane. Among the 15 studied peptides, the peptides exhibiting the least force resistance when passing through the bilayer were found, and the maximum reaction occurred at the boundary of the membrane bilayer entry. We found that the best correlation between the maximum membrane reaction force and the calculated parameters corresponds to the instability index (the correlation coefficient is above 0.9). One of the interesting results of this study is that the 10 residue amyloidogenic peptides and their extended peptides, with nine added residue cell-penetrating peptides and four residue linkers, both with established antimicrobial activity, have the same bilayer resistance force. All calculated data are summarized and posted on the server.     

Glycosyltransferase GLT8D2 Positively Regulates ApoB100 Protein Expression in Hepatocytes

Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation in hepatocytes. Very low density lipoprotein (VLDL) is a major secretory product of the liver that transports endogenously synthesized TG. Disrupted VLDL secretion may contribute to the accumulation of TG in hepatocytes. ApoB100 (apolipoprotein B100) is a glycoprotein and an essential protein component of VLDL. Its glycosylation may affect VLDL assembly and secretion. However, which glycosyltransferase catalyzes apoB100 glycosylation is unknown. In this study, we cloned the GLT8D2 (glycosyltransferase 8 domain containing 2) gene from HepG2 cells and generated a series of plasmids for in vitro studies of its molecular functions. We discovered that GLT8D2 was localized in the ER, interacted with apoB100, and positively regulated the levels of apoB100 protein in HepG2 cells. Based on these results, we propose that GLT8D2 is a glycosyltransferase of apoB100 that regulates apoB100 levels in hepatocytes.     

TP63 Is Significantly Upregulated in Diabetic Kidney

The role of tumor protein 63 (TP63) in regulating insulin receptor substrate 1 (IRS-1) and other downstream signal proteins in diabetes has not been characterized. RNAs extracted from kidneys of diabetic mice (db/db) were sequenced to identify genes that are involved in kidney complications. RNA sequence analysis showed more than 4- to 6-fold increases in TP63 expression in the diabetic mice’s kidneys, compared to wild-type mice at age 10 and 12 months old. In addition, the kidneys from diabetic mice showed significant increases in TP63 mRNA and protein expression compared to WT mice. Mouse proximal tubular cells exposed to high glucose (HG) for 48 h showed significant decreases in IRS-1 expression and increases in TP63, compared to cells grown in normal glucose (NG). When TP63 was downregulated by siRNA, significant increases in IRS-1 and activation of AMP-activated protein kinase (AMPK (p-AMPK-Th¹⁷²)) occurred under NG and HG conditions. Moreover, activation of AMPK by pretreating the cells with AICAR resulted in significant downregulation of TP63 and increased IRS-1 expression. Ad-cDNA-mediated over-expression of tuberin resulted in significantly decreased TP63 levels and upregulation of IRS-1 expression. Furthermore, TP63 knockdown resulted in increased glucose uptake, whereas IRS-1 knockdown resulted in a decrease in the glucose uptake. Altogether, animal and cell culture data showed a potential role of TP63 as a new candidate gene involved in regulating IRS-1 that may be used as a new therapeutic target to prevent kidney complications in diabetes.