Joong-Soo Han

Overexpression of phospholipase D1 in human breast cancer tissues

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. PLD is a major enzyme implicated in important cellular processes, such as cell proliferation. We designed this study to investigate the expression of PLD in human breast carcinomas and non-malignant tissues using RT-PCR, Western blot analysis, immunohistochemistry and an Arf-dependent PLD activity assay. We examined about 550 bp of PCR product and 120 kDa of PLD protein. Our results showed that PLD protein and mRNA levels were overexpressed in 14 of 17 breast cancer tissues. We also observed increased expression by immunohistochemistry and Arf-dependent PLD activity in microsomes of human breast tumors, which correlated well with PLD expression. PLD expression was elevated in human breast tumors compared with normal breast tissues. These results implicate a possible role of PLD in human breast tumorigenesis and suggest that PLD may be useful as a marker for malignant disease in the breast.

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.

Effects of transcription factor activator protein-1 on interleukin-8 expression and enteritis in response to Clostridium difficile toxin A.

Clostridium difficile toxin A causes acute colitis associated with intense infiltration of neutrophils. Although C. difficile toxin A is known to induce nuclear factor-kappaB-mediated chemokine expression in intestinal epithelial cells, little is known about its effect on the regulation of activator protein-1 (AP-1) by mitogen-activated protein kinase (MAPK). In the present study, we investigated whether the MAPK and AP-1 signaling pathway is involved in interleukin (IL)-8 expression and enteric inflammation in response to stimulation with toxin A. Toxin A activated MAPK and AP-1 composed of c-Jun/c-Fos heterodimers in primary intestinal epithelial cells and HT-29 cell lines. Transfection with mutant genes for Ras, c-Jun, p38, or c-Jun N-terminal kinase (JNK) significantly inhibited C. difficile toxin A-induced activation of AP-1 and expression of IL-8 in HT-29 cell lines. Furthermore, the p38 inhibitor SB203580 attenuated toxin A-induced inflammation in vivo in the mouse ileum, evidenced by a significant decrease in neutrophil infiltration, villous destruction, and mucosal congestion. Our results suggest that the Ras/MAPK cascade acts as the upstream signaling for AP-1 activation and IL-8 expression in toxin A-stimulated intestinal epithelial cells and may be involved in the development of enteritis after infection with toxin A-producing C. difficile.

Inhibition of lipopolysaccharide-induced nitric oxide synthesis by nicotine through S6K1-p42/44 MAPK pathway and STAT3 (Ser 727) phosphorylation in Raw 264.7 cells.

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.

Mechanism of silica-induced ROS generation in Rat2 fibroblast cells.

Reactive oxygen species (ROS) play an important role in cell signaling pathway. Previously, we found that silica induced immediate ROS generation and sequential cellular responses such as kinase activation in Rat2 cells as well as an increase of intracellular calcium concentration in A549 cells. However, the detailed mechanism underlying the immediate ROS generation induced by silica in fibroblast cells remains to be elucidated. Therefore, in the present study, we investigated the mechanism of ROS generation by silica within Rat2 fibroblast cells by examining the effects of a diverse group of inhibitors for the enzymes related with signal transduction events. Inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC) and calmodulin (CaM) kinase II effectively suppressed ROS generation in silica-stimulated Rat2 cells, whereas those for protein kinase A and phospholipase A(2) did not. Diphenyleneiodonium chloride (DPI), an inhibitor for NADPH oxidase was also found to be effective in inhibiting silica-induced ROS generation. These results suggest that PTK, PLC, PKC, CaM kinase II, and NADPH oxidase are all involved in signal transduction pathways for ROS generation in silica-stimulated Rat2 cells.

Changes of phospholipase D activity in

The changes of phospholipase D (PLD) activity were investigated during the courses of apoptotic process induced by tumor necrosis factor (TNF)-α or anti-Fas/Apo1 antibody in human premyelocyte HL-60 and murine B cell lymphoma A20 cells. The treatment of recombinant TNF-α 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-α or anti-Fas/Apo1 monoclonal antibody for 6 h. Both TNF-α 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-α 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-α and anti-Fas/Apo1 monoclonal antibody.

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We investigated the mechanism of 8‐Br‐cAMP‐mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8‐Br‐cAMP, maximally at 5 min. To determine whether the effects of 8‐Br‐cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 μM) and GF109203X (1 μM), the PKC inhibitors, or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8‐Br‐cAMP. Furthermore, 8‐Br‐cAMP had no effects on the subcellular distribution of PKC α and PKC βI, confirming no involvement of PKC. 8‐Br‐cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 μM) and transfection of ES cells with dominant negative (DN)‐MEK completely inhibited 8‐Br‐cAMP‐induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8‐Br‐cAMP‐induced PLD activation, we used PKA inhibitor, H89 and Rp‐cAMPs, and transfections of DN‐Src and DN‐Ras. H‐89 and Rp‐cAMPs completely blocked 8‐Br‐cAMP‐mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN‐Src, or DN‐Ras partially inhibited 8‐Br‐cAMP‐induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN‐Src and DN‐Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8‐Br‐cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8‐Br‐cAMP is mediated by Src and Ras, separately.

and anti-Fas/Apo1 monoclonal antibody induced apoptosis in HL-60 and A20 cells

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.

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal 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.

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

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 PLDs 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.