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ABSTRACT: The purpose of this study was to examine the role of phospholipase D1 (PLD1) in basic fibroblast growth factor (bFGF)-induced neurotrophin-3 (NT-3) expression and neurite outgrowth in H19-7 rat hippocampal neuronal progenitor cells. Overexpression of PLD1 increased bFGF-induced NT-3 expression, and dominant-negative-PLD1 or PLD1 siRNA abolished bFGF-induced NT-3 expression and neurite outgrowth. Treatment with bFGF activated the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway, and bFGF-induced NT-3 expression was blocked by a dominant-negative RhoA as well as by a specific Rho-kinase inhibitor (Y27632) and a SAPK/JNK inhibitor (SP600125). Furthermore, bFGF-induced JNK activation was also blocked by Y27632. These results indicate that the RhoA/ROCK/JNK pathway acts as an upstream signaling pathway in bFGF-induced NT-3 expression. Also, phosphatidic acid, the product of PLD, increased NT-3 expression. We found that PLD regulated the RhoA/ROCK/JNK pathway, which then led to Elk-1 transactivation. When Elk-1 activity was blocked by Elk-1 siRNA, bFGF-induced NT-3 expression and neurite outgrowth decreased. NT-3 overexpression increased neurite outgrowth, indicating that NT-3 is important for neurite outgrowth. Taken together, these results suggest that PLD1 is an important regulator of bFGF-induced NT-3 expression and neurite outgrowth, which are mediated by the RhoA/ROCK/JNK pathway via Elk-1 in H19-7 cells.
Molecular Neurobiology 04/2012; 45(3):507-19. · 5.74 Impact Factor
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ABSTRACT: The purpose of this study was to identify the role of phospholipase D (PLD) isozymes in Bcl-2 expression. Overexpression of PLD1 or PLD2 increased Bcl-2 expression and phosphatidic acid (PA), the product of PLDs, also upregulated Bcl-2 expression. Treatment with PA activated the phospholipase A(2) (PLA(2))/G(i)/ERK1/2, RhoA/Rho-associated kinase (ROCK)/p38 MAPK, and Rac1/p38 MAPK pathways. PA-induced phosphorylation of ERK1/2 was attenuated by a PLA(2) inhibitor (mepacrine) and, a G(i) protein inhibitor (pertussis toxin, PTX). On the other hand, p38 MAPK phosphorylation was attenuated by a dominant negative Rac1 and a specific Rho-kinase inhibitor (Y-27632). These results suggest that PLA(2)/G(i) acts at the upstream of ERK1/2, while Rac1 and RhoA/ROCK act upstream of p38 MAPK. We next, tried to determine which transcription factor is involved in PLD-related Bcl-2 expression. When signal transducer and activator of transcription 3 (STAT3) activity was blocked by a STAT3 specific siRNA, PA-induced Bcl-2 expression was remarkably decreased, suggesting that STAT3 is an essential transcription factor linking PLD to Bcl-2 upregulation. Taken together, these findings indicate that PLD acts as an important regulator in Bcl-2 expression by activating STAT3 involving the phosphorylation of Ser727 through the PLA(2)/G(i)/ERK1/2, RhoA/ROCK/p38 MAPK, and Rac1/p38 MAPK pathways.
Biochimica et Biophysica Acta 04/2012; 1823(6):1082-91. · 4.66 Impact Factor
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ABSTRACT: The purpose of the present study was to investigate the role of PLD (phospholipase D) in bFGF (basic fibroblast growth factor)-induced Bcl-2 expression and to examine whether overexpressed Bcl-2 influences neurite outgrowth in immortalized hippocampal progenitor cells (H19-7 cells). We found that Bcl-2 expression was maximally induced by bFGF within 24 h, and that this effect was reduced by inhibiting PLD1 expression with PLD1 small interfering RNA or by overexpressing DN (dominant-negative)-PLD1, whereas PLD1 overexpression markedly induced Bcl-2 expression. bFGF treatment activated Ras, Src, PI3K (phosphoinositide 3-kinase), PLCγ (phospholipase Cγ) and PKCα (protein kinase Cα). Among these molecules, Src and PKCα were not required for Bcl-2 expression. PLD activity was decreased by Ras, PI3K or PLCγ inhibitor, suggesting that PLD1 activation occurred through Ras, PI3K or PLCγ. We found that Ras was the most upstream molecule among these proteins, followed by the PI3K/PLCγ pathway, indicating that bFGF-induced PLD activation took place through the Ras/PI3K/PLCγ pathway. Furthermore, PLD1 was required for activation of JNK (c-Jun N-terminal kinase), which led to activation of STAT3 (signal transducer and activator of transcription 3) and finally Bcl-2 expression. When Bcl-2 was overexpressed, neurite outgrowth was stimulated along with induction of neurotrophic factors such as brain-derived neurotrophic factor and neurotrophin 4/5. In conclusion, PLD1 acts as a downstream effector of bFGF/Ras/PI3K/PLCγ signalling and regulates Bcl-2 expression through JNK/STAT3, which leads to neurite outgrowth in H19-7 cells.
Biochemical Journal 09/2011; 441(1):407-16. · 4.90 Impact Factor
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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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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: 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: Decidualization of human endometrial stromal (ES) cells plays a critical role in successful uterine implantation. Therefore, monitoring of the behavior of human ES cells may provide the clue for early detection of a uterine abnormality such as sterility and abortion. Monitoring of decidualization in vitro cell culture system fundamentally depends on expression of the definite biomarkers. In this study, we tried to uncover novel marker proteins of 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP)-induced decidualization in human ES cells using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Samples were divided into three groups; control human ES cells (n=7), ES cells treated with 8-Br-cAMP (n=7 per each treatment, treated for 3, 6, 9, or 12 days), and cells from which 8-Br-cAMP was withdrawn for 3 days (n=7) or 6 days (n=7) after 8-Br-cAMP treatment for 6 days. Differential expressions between non-decidual control cells and 8-Br-cAMP-induced decidual cells were observed in the peaks of 9787.058 Da, 10115.45 Da, and 24031.25 Da, detected by H4 ProteinChip, and in the peaks of 10833.08 Da, 22440.88 Da, and 32777.38 Da, detected by CM10 ProteinChip. The expression patterns of these decidual markers are expected to provide invaluable information in monitoring cellular development, and further identification of these proteins may hopefully offer precious means for clinical research and therapeutic purposes.
Archives of Pharmacal Research 11/2008; 31(10):1247-55. · 1.59 Impact Factor
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Joong Shin Park,
Kyoung-Jin Oh,
Errol R Norwitz,
Joong-Soo Han, Hye-Jin Choi,
Hyo Suk Seong,
Yoon Dan Kang,
Chan-Wook Park,
Byoung Jae Kim,
Jong Kwan Jun,
Hee Chul Syn
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ABSTRACT: To identify proteomic biomarkers in amniotic fluid (AF) that can distinguish preeclampsia (PE) from chronic hypertension (CHTN) and normotensive controls (CTR).
AF from women with PE, CHTN, and CTR were subjected to proteomic analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.
Proteomic profiling of AF identified 2 biomarkers: peak X (17399.11 Da), which distinguished PE from CTR, and peak Y (28023.34 Da), which distinguished PE and CHTN from CTR. High performance liquid chromatography fractions containing the biomarkers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion. The biomarkers were matched to proapolipoprotein A-I (peak Y) and a functionally obscure peptide, SBBI42 (peak X). Western blot analysis confirmed that AF from PE and CHTN had higher proapolipoprotein A-I levels than CTR.
Proteomic analysis of AF can distinguish PE from CHTN and CTR. The discriminatory proteins were identified as proapolipoprotein A-I and SBBI42.
Reproductive sciences (Thousand Oaks, Calif.) 06/2008; 15(5):457-68. · 2.31 Impact Factor
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ABSTRACT: We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), suggesting that PLA(2) metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA(2) and AA-COX2 pathway.
Journal of Cellular Biochemistry 09/2007; 101(6):1409-22. · 2.87 Impact Factor
