Role of phospholipase D1 in glucose-induced insulin secretion in pancreatic beta cells

As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.

STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells

Phosphatidic acid (PA), the product of a PLD-mediated reaction, is a lipid second messenger that participates in various intracellular signaling events and is known to regulate a growing list of signaling proteins. We found that Bcl-2 was upregulated by PA treatment in HeLa cells. However, how PA upregulates Bcl-2 expression has not yet been studied. In this study, we tried to discover the mechanisms of Bcl-2 up-regulation by PA treatment in HeLa cells. Treatment with PA resulted in significantly increased expression of Bcl-2 in HeLa cells. Moreover, PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of PLA2, but not by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Treatment of 1,2-dipalmitoryl-sn-glycero-3-phosphate (DPPA) also increased Bcl-2 expression. These results indicate that Bcl-2 expression is mediated by lysophosphatidic acid (LPA), not by arachidonic acid (AA). Thereafter, we used MEK1/2 inhibitor, PD98059 to investigate the relationship between ERK1/2 MAPK and PA-induced Bcl-2 expression. PA-induced Bcl-2 expression was decreased when ERK1/2 was inhibited by PD98059. The transcription factor such as STAT3 which is controlled by ERK1/2 MAPK was increased along with Bcl-2 expression when the cells were treated with PA. Furthermore, STAT3 siRNA treatments inhibited PA-induced Bcl-2 expression, suggesting that STAT3 (Ser727) is involved in PA-induced Bcl-2 expression. Taken together, these findings indicate that PA acts as an important mediator for increasing Bcl-2 expression through STAT3 (Ser727) activation via the ERK1/2 MAPK pathway.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Detection of SNP of Phospholipase D1 in Children with Atopic Dermatitis / 소아알레르기및호흡기학회지

BACKGROUND: Phospholipase D (PLD) is a widely distributed enzyme that hydrolyzes phosphatidylcholine, a major phospholipids in the cell membrane, to form phosphatidic acid (PA) which acts by itself as a cellular messenger. PLD can also be transformed by PA phosphohydrolase into diacylglycerol (DAG), which is essential for the activation of protein kinase C (PKC). PLD has been shown to induce the proliferation of T cells and to activate by Der p 1 in peripheral blood mononuclear cells from atopic dermatitis. Single nucleotide polymorphism (SNP) has recently served as a key marker to discover the genetic mechanism of special chronic diseases. METHODS: One hundred eighteen children with atopic dermatitis were recruited, and graded as 23 mild (50) by measuring SCORAD index. Genomic DNA were purified from blood and made into PCR primers attaching GC-Clamp, and 26 exons of PLD were amplified by PCR-DGGE (denaturing gradient gel electrophoresis). RESULTS: Polymorphism was found in four subjects. Of them, three PLD1 cSNP (Exon23: G2658A, T2664A, G2684A) were detected in exon 23 of 26 exons of PLD1. Four cases among 118 subjects had cSNP of G2658A (3.4%), two T2664A cases (1.7%), one G2684A case (0.8%). There were no significant correlations between IgE and detected cSNP. CONCLUSION: Three PLD1 gene cSNPs (G2658A, T2664A, G2684A) were detected in the blood of children with atopic dermatitis. Among them, G2658A polymorphism seems to be correlated to the serum IgE level, but PLD1 cSNP does not appear to contribute to the pathogenic processing of atopic dermatitis.

Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: Involvement of protein kinase C-alpha

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals

Anti-apoptotic role of phospholipase D isozymes in the glutamate-induced cell death

Abstract Phospholipase D (PLD) plays an important role as an effector in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, PLD2 was reported as a necessary intermediate in preventing apoptosis induced by hydrogen peroxide or hypoxia in rat pheochromocytoma (PC12) cells. The data presented here show that both PLD isozymes, PLD1 and PLD2 are also required in attenuating glutamate-induced cell death in PC12 cells. Treatment of PC12 cells with glutamate resulted in induction of apoptosis in these cells, which is accompanied by decreased PLD activity and increased ceramide concentration. Incubation of PC12 cells with exogenous C6-ceramide showed a time-dependent decrease of PLD activity. When cDNAs of PLD1 and PLD2 were transfected into PC12 cells respectively, overexpression of PLD1 or PLD2 resulted in inhibition of glutamate-induced apoptotic cell death. These data indicate that both PLD1 and PLD2 play a protective role against glutamate-induced cell death in PC12 cells.

The Effects of Tibolone in Breast Cancer Cell Lines: The Role of Tibolone in Phospholipase D Signal Transduction Pathway / 한국유방암학회지

PURPOSE: Tibolone is a tissue specific steroid hormone newly recognized as an estrogenic agent for hormone replacement therapy (HRT). The aim of the study is to characterize the basic mechanism of tibolone in the PLD signal transduction pathway of breast cancer cell lines. METHODS: The levels of phospholipase D (PLD), caspase 3 mRNA and protein, and the cell counts were measured in estrogen receptor positive MCF-7 and negative MDA-MB-231 cell lines treated with estradiol, tamoxifen and tibolone and three metabolite forms of tibolone (3 beta-OH-tibolone, delta4 isomer, 3 alpha-OH-tibolone). Multimodality methods such as RT-PCR, immunoblot analysis and in vivo enzyme activity assay were used. RESULTS: The addition of estradiol to MCF-7 cell line resulted in cell proliferation in a time-dependent manner while that of tamoxifen and tibolone showed antiproliferative effects. The addition of tamoxifen or tibolone to MCF-7 and MDA- MB-231 cell lines resulted in the elevation of caspase 3 mRNA levels, indicating the induction of apoptosis. PLD mRNA level was elevated in both cell lines treated with tamoxifen, but decreased in those treated with the various tibolones, except for 3 beta- OH-tibolone. In immunoblot analysis, while MCF-7 cell line treated with tamoxifen showed an increased level of PLD expression, in MDA-MB-231 cell line the expression was decreased. Similar results were observed in the addition of tibolones, which resulted in an increase of PLD expression level in MCF-7 cell line and a decrease in MDA-MB-231 cell line. In vitro PLD activity assay showed decreased activity after estradiol treatment and increased activity after tamoxifen and tibolone treatment in MDA-MB231 cell line. In MCF-7 cell line, among the tibolones only delta4 isomer increased PLD activity. Tiboloneshowed antiproliferative and apoptosis-inducing effects on MCF-7 and MDA-MB-231 cell lines. But its influence on the signal transduction pathway varied slightly between the two cell lines. CONCLUSION: we were able to find the antiestrogenic properties of the estrogenic agent tibolone.

Effect of TRH on Phospholipase D Activity in GH3 Cell / 대한내분비학회지

BACKGROUND: GH3 cells are a well characterized and widely used model used for the in vitro study of growth hormone (GH) secretion. Thyrotropin releasing hormone (TRH) binds to receptors belonging to the family of G protein-coupled receptors, and secrets both GH & prolactin. Phospholipase D (PLD) is an enzyme that hydrolyses phosphatidylcholine to yield phosphatidic acid and choline, and plays important roles in cellular proliferation and hormonal secretion. To elucidate the pathway of the action of TRH in GH3 cells, we investigated the activities of PLC and PLD in GH3 cells treated with TRH or phorbor 12-myristate 13-acetate (PMA). METHODS: GH3 cells were labeled with [3H] myristate, followed by incubation of with 0.3% ethanol, prior to before the addition of the agonists. The total lipids were extracted from the harvested cells following treatment with the agonists. The PLD activity was assessed by measuring [3H] phosphatidylethanol from the [3H] phospholipid using thin layer chromatography. RESULTS: TRH (1 muM) stimulated the PLC activity by 44-fold over that of the control values. TRH (1 microM), mastoparan (5 muM), and PMA (500 muM) for 30 minutes increased PLD activity by 1.9, 1.5 and 2.2 fold, respectively, in comparison to the controls. The PLD activities after 15, 30, 60, 120 and 240 min treatments of TRH (1 microM) were 142%, 170%, 172%, 160% and 115%, respectively. CONCLUSION: These results suggest that TRH stimulates not only the PLC activity, but also the PLD activity in GH3 cells.

Effect of Human Immunoglobulin G in Pneumoconiotic Patients with Pneumonia / 대한산업의학회지

OBJECTIVES: It is well known that pneumoconiotic patients experience impairments of macrophage function, as well as poor penetration of drugs into the fibrotic nodules and the immune system. Resultantly, pneumonia is frequently involved in pneumoconiotic patients and its treatment is not easy. Therefore, we conducted a clinical evaluation of immunoglobulin G which is known to be effective in severe infectious diseases. METHODS: We randomly selected 45 pneumoconiotic patients with pneumonia and classified them into 2 groups. The experimental group (IgG group) was scheduled to receive antibiotics and IgG (5 g I.V./day for 7 days). The control group was treated with antibiotics alone. Sputum gram stain (counts of WBCs and microorganisms), body temperature, arterial oxygen tension, and counts of peripheral venous blood leukocytes and band neutrophils were used as markers to assess the response effect therapy at time periods of 0, 2, 4, 6, and 8 days after completion of therapy. We compared the clinical scores between the two groups. RESULTS: The experimental IgG treated group was composed of 27 patients, and the control group comprised 18 patients. There was no statistical differences between two groups in terms of age, pneumoconiotic profusion, impairment degree of pulmonary function, or frequency of pathogen isolation in the sputum before medication. The experimental IgG treated group showed lower clinical scores as compared with the control group (p=0.083). CONCLUSIONS: These results suggest that IgG infusion with antibiotics will have an effect on pneumonia therapy in pneumoconiosis patients that are under 60 years and exhibit simple pneumoconiosis.

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidy-lcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.

D60-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.

D60-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.

Molecular cloning and sequencing of rat Cdc42 GTPase cDNA

Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.

Major house dust mite allergen, Der p I, activates phospholipase D in human peripheral blood mononuclear cells from allergic patients: involvement of protein kinase C

The major house-dust-mite allergen, Der p I, stimulates the phospholipase D (PLD) in peripheral blood mononuclear cells (PBMC) from allergic patients with maximal responses after 30 min exposure. At 30 min, Der p I stimulated PLD activity by 1.4-fold in mild, 1.6-fold in moderate and 2-fold in severe allergic patients over control values (p < 0.05). When the cells were pretreated for 24 h with phorbol myristate acetate to down-regulate protein kinase C (PKC), PLD stimulation by Der p I was largely abolished. These results indicate that in PBMC from allergic patients, Der p I can stimulate PLD activity, and that PKC activation is involved in this stimulation.

Analysis of trimethylsilyl derivatization products of phosphatidylethanol by gas chromatography-mass spectrometry

For the detection of rare phospholipid, phosphatidylethanol (PEt), GC-MS analysis method was adopted for the detection of derivatization products of PEt by N,O-bis (trimethylsilyl) trifluroacetamide (BSTFA). A re-structured molecule derived from PEt, ethyl bis (trimethylsilyl)-phosphate was found from search of Wiley database. This molecule can be used as a marker for PEt analysis. The molecular formula was C8H23O4PSi2 and weight of the formula was 270.09.

Effect of ceramide on apoptosis and phospholipase D activity in FRTL-5 thyroid cells

Ceramide, a product of sphingomyelin hydrolysis, is now recognized as an intracellular lipid messenger, which mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. Recently, ceramide has been implicated in the regulation of phospholipase D (PLD). In this study, we examined the effects of ceramide on the activity and mRNA level of PLD during apoptotic process in FRTL-5 thyroid cells. C2-ceramide (N-acetyl sphingosine) induced apoptosis in FRTL-5 thyroid cells. Fluorescent staining showed that ceramide induced the typical features of apoptosis including condensed or fragmented nuclei. DNA fragmentation was also observed by agarose gel electrophoresis. Flow cytometric cell cycle analysis showed more clearly that ceramide induced apoptotic cell death in FRTL-5 thyroid cells. The treatment of FRTL-5 thyroid cells with thyroid-stimulating hormone (TSH) resulted in an increased PLD activity in a dose- and time-dependent manner. However, the TSH-induced increase in PLD activity was down-regulated within 2 h after ceramide treatment. Furthermore, the levels of PLD mRNA were found to be decreased throughout apoptotic process as inferred by reverse transcription-polymerase chain reaction. However, the decreases in PLD mRNA levels were not correlated with those in PLD activities after ceramide treatment. Taken together, these data suggest that ceramide inhibits the PLD activity in an early apoptotic phase and down-regulation of the levels of PLD mRNA may be implicated in apoptotic process in FRTL-5 thyroid cells.

Changes of phospholipase D activity in TNF-alpha and anti-Fas/Apo1 monoclonal antibody induced apoptosis in HL-60 and A20 cells

The changes of phospholipase D (PLD) activity were investigated during the courses of apoptotic process induced by tumor necrosis factor (TNF)-alpha or anti-Fas/Apo1 antibody in human premyelocyte HL-60 and murine B cell lymphoma A20 cells. The treatment of recombinant TNF-alpha to HL-60 cells resulted in the increased PLD activity as determined by the phosphatidylethanol formation in the presence of 1% ethanol. The enhancement of PLD activity was also observed in the anti-Fas/Apo1 monoclonal antibody-treated A20 cells. However, the activity of PLD was maximized when HL-60 and A20 cells were treated with either TNF-alpha or anti-Fas/Apo1 monoclonal antibody for 6 h. Both TNF-alpha and anti-Fas/Apo1 monoclonal antibody increased PLD activity in a dose-dependent manner up to 200 U/ml and 200 ng/ml, respectively. When the intracellular activity of protein kinase C (PKC) was interrupted by treatment of calphostin-C, both the PLD activation and the apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody appeared to be inhibited. Since PKC is reported to activate PLD, the results indicate that the intracellular signaling cascade via PLD may play a role in the induction of apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody.

Expression of a cDNA for hippocalcin from rat brain

A hippocalcin cDNA from rat brain cDNA library was amplified by polymerase chain reation(PCR) and cloned using TA Cloning technique. For this PCR cloning, 29mer and 28mer oligonucleotide primers containing BamHl and EcoRl sites at the 5' end and 3' end, respectively were used. The nucleotide sequence of hippocalcin cDNA c1one was determined, and the complete amino acid sequence was deduced. Recombinant clone contained a cDNA insert of 610 base pairs with 582 nucleotides of open reading frame including the temination codon, 23 nucleotide of 5'-untranslated region, and 5nucleotides of 3'-nutran,slated region. The open reading frame encoded a polypepetid comprising 193 amino acids with molecular weight of 22kDa. The cDNA insert was subcloned into pVLI393 Baculovirus transfer vector. The recombinant hippocalcin was expressed in insect cell(Sf9 cell) using expression vector pVL1393. The hippocalcin expressed was purified as a single band on polyacrylamide gel electrophoresis following hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. Molecular size of rat brain hippocalcin protein expressed in this system was estimated to be 22kDa. Myristoylated hippocalcin migrated faster than nonmyristoyated form on SDS-polyacrylamide gel. Less than 10% of total hippocalcin expressed was myristoylated in this baculovirus expression system.

The Combined Therapeutic Effect of Drugs in Experimental Acute Focal Cerebral Ischemia

The study was designed to examine the effects of pretreatment with mannitol, methyl prednisolone and nimodipine on the acute focal cerebral ischemia in the cats of occlusion of the proximal part of the middle cerebral artery via the postorbital approach. The energy metabolisms of the brain was measured utilizing the high liquid performance chromatography in the brain tissues of cats. The experimental animals were seperated into 3 groups. group I: the sham control group. group II: the recirculation group. group III: the treatment group. There were significant increase in the ATP, GTP, UTP and E.C. levels in focal ischemic cerebral tissues of the treatment group when compared with the recirculation group. It is suggested that pretreatment with the combination of these drugs may prevent the ischemic damage from the acute focal cerebral ischemia by the maintenance of high energy metabolites. However further studies should determine the synergistic pharmacologic mechanisms in this therapeutic strategy.