Shin-Young Park

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.

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 suppresses taxotere-induced cell death in stomach cancer cells

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.

Effect of sildenafil citrate on interleukin-1β-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-1β-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1β stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1β-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-1β 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-1β-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1β-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.

Phospholipase D1 activation through Src and Ras is involved in basic fibroblast growth factor‐induced neurite outgrowth of H19‐7 cells

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‐γ) 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‐γ 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. J. Cell. Biochem. 101: 221–234, 2007.

A Novel Role of Hippocalcin in bFGF-Induced Neurite Outgrowth of H19-7 Cells

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–γ (PLC‐γ) and an increased level of intracellular Ca2+. Hippocalcin expression by bFGF stimulation was fully blocked by both the PLC‐γ inhibitor U73122 and BAPTA‐AM, a chelator of intracellular Ca2+, suggesting that hippocalcin expression by bFGF is dependent on PLC‐γ 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‐γ activation, which leads to neurite outgrowth.

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.

GENOMIC DNA SEQUENCE OF A RICE GENE CODING FOR A PULLULANASE-TYPE OF STARCH DEBRANCHING ENZYME

A genomic DNA containing a rice (Oryza sativa L., cv. Norin-8) gene coding for a pullulanase-type starch debranching enzyme (EC 3.2.1. 41) was sequenced (EMBL/GenBank/DDBJ accession number AB012915). Along the 15, 248 bp DNA, the pullulanase gene is split into 26 exons. The four pullulanase consensus regions are positioned in the middle portion of the sequence and are separated by long introns and 1-3 exons. Comparison of the rice cv. Norin-8 pullulanase genomic structure with that of barley pullulanase (limit dextrinase) (F. Lok et al., EMBL/GenBank/DDBJ accession number AF022725) indicates that most of the pullulanase exons are highly conserved. Alignment of the nucleotide bases of rice exon 8 with those of barley exon 8-intron 8-exon 9 fragment suggests that the 85 bp internal sequence of rice exon 8 was originally an intron, a possibility further indicated by the absence in barley and spinach (A. Renz et al., EMBL/GenBank/DDBJ accession number X83969) pullulanases of amino acid residues encoded by the 85 bp fragment.

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.

Nicotine Inhibits bFGF-induced Neurite Outgrowth through Suppression of NO Synthesis in H19-7 Cells

NO (Nitric oxide) has been known as a biological signaling molecule that can function as a beneficial agent in physiologically essential functions such as differentiation or neurotransmission. In this study, we elucidated how nicotine inhibits neuronal differentiation induced by the basic fibroblast growth factor (bFGF) in hippocampal cell line, H19-7 cells, because nicotine is one of the key neuroregulatory components. Treatment of H19-7 cells with bFGF increased NO production through upregulated iNOS/nNOS expression, and also increased expressions of neuronal markers such as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and Neuro-D. Pretreatment of the cells with nicotine decreased iNOS promoter activity as well as iNOS/nNOS expression induced by bFGF, resulting in decreased NO production. Nicotine also suppressed expressions of BDNF, NT3 and Neuro-D, resulting in decreased bFGF-induced neurite outgrowth. These results indicate that nicotine inhibits bFGF-induced neuronal differentiation in H19-7 cells through inhibition of NO formation by suppressing iNOS/nNOS expressions.