Hye-Jin Choi

Identification of Proteomic Biomarkers of Preeclampsia in Amniotic Fluid Using SELDI-TOF Mass Spectrometry

Objective: To identify proteomic biomarkers in amniotic fluid (AF) that can distinguish preeclampsia (PE) from chronic hypertension (CHTN) and normotensive controls (CTR). Methods: AF from women with PE, CHTN, and CTR were subjected to proteomic analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Results: 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. Conclusion: Proteomic analysis of AF can distinguish PE from CHTN and CTR. The discriminatory proteins were identified as proapolipoprotein A-I and SBBI42.

Role of Phospholipase D2 in Anti-Apoptotic Signaling Through Increased Expressions of Bcl-2 and Bcl-xL

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 A2 (PLA2), suggesting that PLA2 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 PLA2 and AA‐COX2 pathway. J. Cell. Biochem. 101: 1409–1422, 2007.

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.

Overexpression of phospholipase D enhances Bcl-2 expression by activating STAT3 through independent activation of ERK and p38MAPK in HeLa cells

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.

Phospholipase D2 acts as an important regulator in LPS-induced nitric oxide synthesis in Raw 264.7 cells.

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.

Leptin Increases TNF-α Expression and Production through Phospholipase D1 in Raw 264.7 Cells

Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.

The TSP motif in AP180 inhibits phospholipase D1 activity resulting in increased efficacy of anticancer drug via its direct binding to carboxyl terminal of phospholipase D1

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.

Phospholipase D1 is an important regulator of bFGF-induced neurotrophin-3 expression and neurite outgrowth in H19-7 cells.

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.

The TLR4-associated phospholipase D1 activation is crucial for Der f 2-induced IL-13 production

House dust mites (HDMs) are the most important source of indoor aeroallergens that contribute to the rising incidence of allergic diseases such as allergic asthma. The major HDM, Der f 2, induces inflammatory cytokine expression. Little is known about the signaling pathway involved.

TLR4-mediated IRAK1 activation induces TNF-α expression via JNK-dependent NF-κB activation in human bronchial epithelial cells

The purpose of this study was to identify the mechanism of lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF)-α in BEAS-2B. Toll-like receptor (TLR)4-specific siRNA was found to completely abolish the LPS-induced expression of MyD88 and TNF-α. There was enhanced binding of MyD88 with IRAK1 following LPS treatment, and MyD88- or IRAK1-specific siRNAs decreased the expression of TNF-α. In addition, IRAK1 siRNA downregulated the phosphorylation of PKCα, demonstrating that PKCα is a downstream effector of IRAK1. Inhibition of PKCα completely blocked the activation of AKT, whereas inhibition of AKT with a PI3K inhibitor prevented the LPS-induced expression of TNF-α. We found that AKT activated JNK, which then stimulated phosphorylation of Iκ-Bα, resulting in NF-κB activation. As expected, inhibition of NF-κB completely inhibited the expression of TNF-α. Taken together, our results suggest that LPS induces TNF-α expression by activating NF-κB via the PKCα/PI3K/AKT/JNK pathway, which is in turn dependent on MyD88/IRAK1.