Byoung Dae Lee

Sensitization of Epidermal Growth Factor-induced Signaling by Bradykinin Is Mediated by c-Src IMPLICATIONS FOR A ROLE OF LIPID MICRODOMAINS

Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) γ1 to the rafts. Depletion of cholesterol by methyl-β-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCγ1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.

Lysophosphatidylcholine Activates Adipocyte Glucose Uptake and Lowers Blood Glucose Levels in Murine Models of Diabetes

Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cδ inhibitor) and by the adenoviral expression of dominant negative protein kinase Cδ. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.

Phospholipase D2 Activity Suppresses Hydrogen Peroxide‐Induced Apoptosis in PC12 Cells

Abstract: Phospholipase D (PLD) plays an important role as an effector in the membrane lipid‐mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H2O2)‐induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H2O2 resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H2O2‐induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1‐butanol. Expression of PLD2, but not of PLD1, correlated with increased H2O2‐induced PLD activity in a concentration‐ and time‐dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H2O2‐induced apoptosis in PC12 cells. Expression of PLD2 lipase‐inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H2O2‐induced cleavage and activation of caspase‐3. Taken together, the results suggest that PLD2 activity is specifically up‐regulated by H2O2 in PC12 cells and that it plays a suppressive role in H2O2‐induced apoptosis.

Localization of phospholipase C-γ1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation

Upon epidermal growth factor treatment, phospholipase C‐γ1 (PLC‐γ1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC‐γ1 and its tyrosine phosphorylation are localized in caveolae by caveolin‐enriched low‐density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl‐β‐cyclodextrin (MβCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC‐γ1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MβCD shows no effect on tyrosine phosphorylation level of PLC‐γ1. Our findings suggest that, for proper signaling, PLC‐γ1 phosphorylation has to occur at PtdInsP2‐enriched sites.

Pituitary Adenylate Cyclase-Activating Polypeptide 27 Is a Functional Ligand for Formyl Peptide Receptor-Like 1

Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophile leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.

Epidermal growth factor increases insulin secretion and lowers blood glucose in diabetic mice

Epidermal growth factor (EGF) is synthesized in the pancreas and diabetic animals have low levels of EGF. However, the role of EGF in regulating the major function of the pancreas, insulin secretion, has not been studied. Here, we show that EGF rapidly increased insulin secretion in mouse pancreatic islets, as well as in a pancreatic β‐cell line. These events were dependent on a Ca2+ influx and phospholipase D (PLD) activity, particularly PLD2, as determined using pharmacological blockers and molecular manipulations such as over‐expression and siRNA of PLD isozymes. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in normal and diabetic mice. Here, for the first time, we provide evidence that EGF is a novel secretagogue that regulates plasma glucose levels and a candidate for the development of therapeutics for diabetes.

Bradykinin activates phospholipase D2 via protein kinase Cδ in PC12 cells

Abstract Bradykinin (BK) activates phospholipase D (PLD) and induces several responses such as catecholamine secretion, collapse of growth cones, and gene expression in PC12 pheochromocytoma cells. Although two distinct PLD isozymes, PLD1 and PLD2, have been cloned from mammalian cells, the regulatory mechanism for each PLD isozyme by BK is not clear. In our present study, we investigated the activation mechanism of PLD2 by BK in PLD2-overexpressing PC12 cells. BK stimulated PLD2 activity in a concentration-dependent manner within 1 min and this activation was inhibited by pretreatment of the cells with protein kinase C (PKC) inhibitor. PKCα and PKCδ translocated from cytosol to membrane upon BK treatment, and rottlerin potently inhibited the activation of PLD2 by BK. These results suggest that BK activates PLD2 via PKCδ in PC12 cells.

Hydrogen peroxide-induced phospholipase D2 activation in lymphocytic leukemic L1210 cells.

Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholipase D (PLD). However, it was still unclear how this activation occurs and what the molecular identity of the H2O2‐stimulated PLD isozyme is. This study shows that H2O2 potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD2. In addition, H2O2 increased PLD activity only in PLD2‐transfected COS‐7 cells and not in PLD1‐transfected cells. This suggests that PLD2 is selectively activated by H2O2. Depletion of extracellular Ca2+ with EGTA completely blocked the H2O2‐induced PLD activation, indicating that Ca2+ influx is required. Moreover, pretreatment of the cells with the protein kinase C (PKC) inhibitors GF‐109203X and RO‐31‐8220 and down‐regulation of PKCct by prolonged treatment with 4β‐phorbol 12‐myristate 13‐acetate inhibited the H2O2‐stimulated PLD2 activity, which points to the involvement of PKCα. Based on these new findings we suggest that PLD2 activity is specifically up‐regulated by H2O2 and that the H2O2‐induced PLD2 activation is mediated by Ca2+ influx and PKCα activation. J. Leukoc. Biol. 67: 630–636; 2000.

Leumorphin has an anti-apoptotic effect by activating epidermal growth factor receptor kinase in rat pheochromocytoma PC12 cells.

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, β‐neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3‐kinase and mitogen‐activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor‐binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition‐binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src‐dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation‐induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor‐mediated activation of the phosphatidylinositol 3‐kinase and mitogen‐activated protein kinase pathways, which occur independent of the kappa opioid receptor.