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ABSTRACT: Phospholipase D (PLD) has two isoforms, PLD1 and PLD2. Both isoforms are possible candidates for the development of anticancer drugs, since PLDs in several cancer cells act as survival factors. The aim of this study was to elucidate the inhibitory mechanism of PLD1 by AP180 in human cancer cells. Transfection of the human AP180 (hAP180) gene markedly inhibited phobol-12-myristate 13-acetate-induced PLD activity resulting in exacerbation of anticancer drug-induced cell death. Experiments using deletion mutants of hAP180 showed that three amino acids (Thr312-Pro314) are critical for inhibition of PLD1 activity by binding directly to PLD1, and, of these, Ser313 was the most important residue for both binding to and inhibiting PLD1. However, this inhibitory relationship did not exist between hAP180 and PLD2. In addition, the C-terminal region of PLD1 is important for the interaction with hAP180. These results indicated that Thr312-Pro314 (especially Ser313 as a phosphorylation residue) of hAP180 can regulate hPLD1 activity through binding with the C-terminal region of PLD1.
Cancer letters 03/2011; 302(2):144-54. · 4.86 Impact Factor
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Myoung Sook Han,
Yu-Mi Lim,
Wenying Quan,
Jung Ran Kim,
Kun Wook Chung,
Mira Kang,
Sunshin Kim,
Sun Young Park,
Joong-Soo Han, Shin-Young Park,
Hyae Gyeong Cheon,
Sang Dal Rhee,
Tae-Sik Park,
Myung-Shik Lee
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ABSTRACT: The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα(i), reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.
The Journal of Lipid Research 03/2011; 52(6):1234-46. · 5.56 Impact Factor
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ABSTRACT: 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.
Experimental and Molecular Medicine 05/2010; 42(6):456-64. · 2.48 Impact Factor
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ABSTRACT: The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophage cell line, Raw 264.7 cells. When Raw 264.7 cells were stimulated with LPS, the expressions of PLDs were increased. Thus, to investigate the role of PLD in NO synthesis, we transfected PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, only PLD2 overexpression, but not that of PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the S6K1-p42/44 MAPK-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. Knockdown of PLD2 with siRNA also decreased phosphorylation of S6K1, p42/44 MAPK and STAT3 induced by LPS. Furthermore, we found that STAT3 bound with the iNOS promoter, and their binding was mediated by PLD2. Taken together, our results demonstrate the importance of PLD2 for LPS-induced NO synthesis in Raw 264.7 cells with involvement of the S6K1-p42/44 MAPK-STAT3 pathway.
Cellular signalling 12/2009; 22(4):619-28. · 4.09 Impact Factor
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ABSTRACT: The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Der f 2-induced interleukin (IL)-13 production. The major house dust mite allergen, Der f 2, increased PLD activity in human bronchial epithelial cells (BEAS-2B), and dominant negative PLD1 or PLD1 siRNA decreased Der f 2-induced IL-13 expression and production. Treatment of Der f 2 activated the phospholipase Cgamma (PLCgamma)/protein kinase Calpha (PKCalpha)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCgamma inhibitors (U73122 and PAO), PKCalpha inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCgamma, PKCalpha, and p38 MAPK act as upstream activators of PLD in Der f 2-treated BEAS-2B cells. Furthermore, expression and production of IL-13 increased by Der f 2 were also blocked by inhibition of PLCgamma, PKCalpha, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCgamma/PKCalpha/p38 MAPK pathway. We found that activating transcription factor-2 (ATF-2) was activated by Der f 2 in BEAS-2B cells and activation of ATF-2 was controlled by PLD1. When ATF-2 activity was blocked with ATF-2 siRNA, Der f 2-induced IL-13 expression and production were decreased. Thus, ATF-2 might be one of the transcriptional factors for the expression of IL-13 in Der f 2-treated BEAS-2B cells. Taken together, PLD1 acts as an important regulator in Der f 2-induced expression and production of IL-13 through activation of ATF-2 in BEAS-2B cells.
Journal of Biological Chemistry 07/2009; 284(30):20099-110. · 4.77 Impact Factor
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ABSTRACT: 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.
Experimental and Molecular Medicine 03/2009; 41(2):94-101. · 2.48 Impact Factor
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ABSTRACT: Lipopolysaccharide (LPS) has been known to produce inflammatory modulators such as tumor necrosis factor alpha (TNF-alpha) or nitric oxide (NO). In this study, we examined the effects of nicotine on LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophages. LPS-induced NO synthesis and iNOS expression were significantly decreased by nicotine. To investigate the signaling mechanism of nicotine induced suppression of NO synthesis and iNOS expression induced by LPS, we focused on the possible roles of p42/44 MAPK, S6K1, and signal transducers and activators of transcription 3 (STAT3) signaling. LPS is known to activate p42/44 MAPK and S6K1, which in turn activates STAT3 to induce inflammatory regulators. Pretreatment of cells with nicotine blocked LPS-induced p42/44 MAPK and S6K1 as well as iNOS promoter activity. Furthermore, we found that LPS-induced phosphorylation of STAT3 at serine 727 is mediated by S6K1-p42/44 MAPK pathway, and this STAT3 phosphorylation was also blocked by nicotine. We also found that downregulation of STAT3 using STAT3 siRNA resulted in suppression of the NO synthesis and iNOS expression. Taken together, our results suggest that nicotine inhibits LPS-induced NO synthesis through suppression of S6K1-p42/44 MAPK pathway and phosphorylation of STAT3 in Raw 264.7 cells.
Cytokine 09/2008; 44(1):126-34. · 3.02 Impact Factor
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ABSTRACT: The purpose of this study was to identify the effect of sildenafil citrate on IL-1beta-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1? stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1beta-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-1beta 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-1beta-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1?-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.
Experimental and Molecular Medicine 07/2008; 40(3):286-93. · 2.48 Impact Factor
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ABSTRACT: Hippocalcin is a Ca2+-binding protein that is expressed mainly in pyramidal nerve cells of the hippocampus. However, its functions and mechanism in the brain remain unclear. To elucidate the role of hippocalcin, we used a conditionally immortalized hippocampal cell line (H19-7) and showed that bFGF treatment increased the expression of hippocalcin during bFGF-induced neurite outgrowth of H19-7 cells. Overexpression of hippocalcin dramatically elongated neurites and increased the expression of basic helix-loop-helix transcription factor, that is, NeuroD without bFGF stimulation. Treatment of the cells with hippocalcin siRNA completely blocked bFGF-induced neurite outgrowth and NeuroD expression. bFGF stimulation resulted in activation of phospholipase C-gamma (PLC-gamma) and an increased level of intracellular Ca2+. Hippocalcin expression by bFGF stimulation was fully blocked by both the PLC-gamma inhibitor U73122 and BAPTA-AM, a chelator of intracellular Ca2+, suggesting that hippocalcin expression by bFGF is dependent on PLC-gamma and Ca2+. Moreover, both U73122 and BAPTA-AM completely blocked bFGF-induced neurite outgrowth and NeuroD expression. Taken together, these results suggest for the first time that bFGF induces hippocalcin expression in H19-7 cells through PLC-gamma activation, which leads to neurite outgrowth.
Journal of Neuroscience Research 06/2008; 86(7):1557-65. · 2.74 Impact Factor
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ABSTRACT: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase 3 was inhibited after PLD's transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU 484 cells. On top of this, PA -- the product of PLDs, also resulted in upregulation of Bcl-2 in SNU 484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA(2), but not with PAP, during taxotere-induced apoptosis in SNU 484 cells.
Biochimica et Biophysica Acta 06/2008; 1783(5):912-23. · 4.66 Impact Factor
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ABSTRACT: Phospholipase D (PLD) is implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. We found that PLD1 is activated when basic fibroblast growth factor (bFGF) stimulates neurite outgrowth of an immortalized hippocampal cell line (H19-7). Overexpression of PLD1 in H19-7 cells dramatically elongated bFGF-induced neurite outgrowth and increased PLD activity. Transfection of DN-rPLD1 blocked bFGF-induced PLD activation and completely inhibited neurite outgrowth induced by bFGF, suggesting that PLD1 activation is important in bFGF-induced neurite outgrowth of H19-7 cells. PLD activation and neurite outgrowth induced by bFGF was dependent on phospholipase C gamma (PLC-gamma) and Ca2+, but not protein kinase C (PKC). Furthermore, inhibition of Src and Ras partially blocked bFGF-induced PLD activation and neurite outgrowth, respectively. Coinhibition of Src and Ras completely blocked bFGF-induced PLD activation, suggesting that Src and Ras independently regulate PLD1 activation. Interestingly, bFGF-induced PLD activation and neurite outgrowth did not require ERK1/2 activated by Ras. Taken together, this study demonstrates that bFGF activates PLD1 through PLC-gamma activation, which leads to neurite outgrowth in H19-7 cells. Furthermore, our results show that PLD1 activation by bFGF is regulated by Src and Ras independently.
Journal of Cellular Biochemistry 06/2007; 101(1):221-34. · 2.87 Impact Factor
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ABSTRACT: Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.
Biochemical and Biophysical Research Communications 05/2005; 329(3):804-11. · 2.48 Impact Factor
