Hyeok-Yil Kwon

β-arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser83 and microglia chemotaxis

Microglia play crucial roles in increased inflammation in the central nervous system upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y12 receptor. In this study, we examined the role of ERK1/2 activation in ADP‐induced microglia chemotaxis. ADP stimulation increases the phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and paxillin phosphorylation at Tyr31 and Ser83. Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser83 and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion (FA) formation with green fluorescent protein‐paxillin revealed that the disassembly of FAs in U0126‐treated cells was significantly impaired. Depletion of β‐Arrestin 2 (β‐Arr2) with short hairpin RNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser83, indicating that β‐Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β‐Arr2 were translocated and co‐localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr31 by c‐Src appears to be involved in adhesion formation upon ADP stimulation while Ser83 required for adhesion disassembly.

Adjuvant effect of liposome-encapsulated natural phosphodiester CpG-DNA.

Immunostimulatory CpG-DNA targeting TLR9 is one of the most extensively evaluated vaccine adjuvants. Previously, we found that a particular form of natural phosphodiester bond CpG-DNA (PO-ODN) encapsulated in a phosphatidyl-Β-oleoyl- γ-palmitoyl ethanolamine (DOPE) : cholesterol hemisuccinate (CHEMS) (1 : 1 ratio) complex (Lipoplex(O)) is a potent adjuvant. Complexes containing peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Here, we showed that IL-12 production was increased in bone marrow derived dendritic cells in a CpG sequence-dependent manner when PO-ODN was encapsulated in Lipoplex(O), DOTAP or lipofectamine. However, the effects of Lipoplex(O) surpassed those of PO-ODN encapsulated in DOTAP or lipofectamine and also other various forms of liposome-encapsulated CpG-DNA in terms of potency for protein antigen-specific IgG production and Th1- associated IgG2a production. Therefore, Lipoplex(O) may have a unique potent immunoadjuvant activity which can be useful for various applications involving protein antigens as well as peptides.

Involvement of small GTPase RhoA in the regulation of superoxide production in BV2 cells in response to fibrillar Aβ peptides.

Fibrillar amyloid-beta (fAβ) peptide causes neuronal cell death, which is known as Alzheimers disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAβ. We observed that fAβ rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAβ1-42 and fAβ25-35 induced superoxide production in BV2 cells. fAβ25-35 produced superoxide, although fAβ25-35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAβ does not seem to be dependent on phagocytosis of fAβ. Herein we studied how fAβ produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAβ. Furthermore, fAβ elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47(PHOX). Moreover, peritoneal macrophages from p47(PHOX) (-/-) knockout mouse could not produce superoxide in response to fAβ. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAβ through phosphorylation of p47(PHOX) in microglial BV2 cells.

Effects of lipopolysaccharide and CpG-DNA on burn-induced skin injury

Destruction of the skin barrier by thermal injury induces microbial invasion, which can lead to the development of systemic infection and septic shock. Microbial pathogens possess pathogen-associated molecular patterns (PAMPs), which are recognized by conserved receptors. To understand the role of PAMPs in thermal injury-induced mice, LPS or CpG-DNA were topically applied to dorsal skin after thermal injury. We observed an increase in the number of inflammatory cell infiltrates as well as thickening in the dermis upon treatment with LPS or CpG-DNA. We also found that expression of IL-1β, MIP-2, and RANTES induced by thermal injury was enhanced by LPS or CpG-DNA. In addition, the proportions of CD4(+) and CD8(+) T cells in the spleen and lymph nodes were altered by LPS or CpG-DNA. These results provide important information concerning PAMPs-induced inflammation upon thermal injury and provide a basis for studying the role of PAMPs in thermal injury-induced complications.

Activity-dependent variations in conduction velocity of C fibers of rat sciatic nerve.

Changes in the conduction velocity and subsequent conduction block were characterized following impulse activity in single C fibers of rat sciatic nerves. C fibers which had the same resting conduction velocities often exhibited quite different profiles of the activity-dependent latency change and/or conduction block following impulses. The results imply underlying variation among C fibers in the activity-dependent excitability changes, especially in the build-up and recovery of the hypoexcitable phases.