Tat-CIAPIN1 protein prevents against cytokine-induced cytotoxicity in pancreatic RINm5F ß-cells.

Cytokines activate inflammatory signals and are major mediators in progressive ß-cell damage, which leads to type 1 diabetes mellitus. We recently showed that the cell-permeable Tat-CIAPIN1 fusion protein inhibits neuronal cell death induced by oxidative stress. However, how the Tat-CIAPIN1 protein affects cytokine-induced ß-cell damage has not been investigated yet. Thus, we assessed whether the Tat-CIAPIN1 protein can protect RINm5F ß-cells against cytokine-induced cytotoxicity. In cytokine-exposed RINm5F ß-cells, the transduced Tat-CIAPIN1 protein elevated cell survivals and reduced reactive oxygen species (ROS) and DNA fragmentation levels. The Tat-CIAPIN1 protein reduced mitogen-activated protein kinases (MAPKs) and NF-κB activation levels and elevated Bcl-2 protein, whereas Bax and cleaved Caspase-3 proteins were decreased by this fusion protein. Thus, the protection of RINm5F ß-cells by the Tat-CIAPIN1 protein against cytokine-induced cytotoxicity can suggest that the Tat-CIAPIN1 protein might be used as a therapeutic inhibitor against RINm5F ß-cell damage. [BMB Reports 2021; 54(9): 458-463].

MicroRNA-22 negatively regulates LPS-induced inflammatory responses by targeting HDAC6 in macrophages.

Dysregulation of histone deacetylase 6 (HDAC6) can lead to the pathologic states and result in the development of various diseases including cancers and inflammatory diseases. The objective of this study was to elucidate the regulatory role of microRNA-22 (miR-22) in HDAC6-mediated expression of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated macrophages. LPS stimulation induced HDAC6 expression, but suppressed miR-22 expression in macrophages, suggesting possible correlation between HDAC6 and miR-22. Luciferase reporter assays revealed that 3'UTR of HDAC6 was a bona fide target site of miR-22. Transfection of miR-22 mimic significantly inhibited LPS-induced HDAC6 expression, while miR-22 inhibitor further increased LPS-induced HDAC6 expression. LPS-induced activation of NF-κB and AP-1 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. LPS-induced expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. Taken together, these data provide evidence that miR-22 can downregulate LPS-induced expression of proinflammatory cytokines via suppression of NF-κB and AP-1 axis by targeting HDAC6 in macrophages. [BMB Reports 2020; 53(4): 223-228].

PEP-1-paraoxonase 1 fusion protein prevents cytokine-induced cell destruction and impaired insulin secretion in rat insulinoma cells.

Pancreatic beta cell destruction and dysfunction induced by cytokines is a major cause of type 1 diabetes. Paraoxonase 1 (PON1), an arylesterase with antioxidant activity, has been shown to play an important role in preventing the development of diabetes in transgenic mice. However, no studies have examined the anti-diabetic effect of PON1 delivered to beta cells using protein transduction. In this study, we expressed the cell-permeable PON1 fused with PEP-1 protein transduction domain (PEP-1-PON1) to investigate whether transduced PEP-1-PON1 protects beta cells against cytokine-induced cytotoxicity. PEP-1-PON1 was effectively delivered to INS-1 cells and prevented cytokine-induced cell destruction in a dose-dependent manner. Transduced PEP-1-PON1 significantly reduced the levels of reactive oxygen species (ROS) and nitric oxide (NO), DNA fragmentation, and expression of inflammatory mediators, endoplasmic reticulum (ER) stress proteins, and apoptosis-related proteins in cytokine-treated cells. Moreover, transduced PEP-1-PON1 restored the decrease in basal and glucose-stimulated insulin secretion induced by cytokines. These data indicate that PEP-1-PON1 protects beta cells from cytokine-induced cytotoxicity by alleviating oxidative/nitrosative stress, ER stress, and inflammation. Thus, PEP-1-mediated PON1 transduction might be an effective method to reduce the extent of destruction and dysfunction of pancreatic beta cells in autoimmune diabetes. [BMB Reports 2018; 51(10): 539-544].

Protective effects of Tat-DJ-1 protein against streptozotocin-induced diabetes in a mice model.

A major feature of type 1 diabetes mellitus (T1DM) is hyperglycemia and dysfunction of pancreatic ß-cells. In a previous study, we have shown that Tat-DJ-1 protein inhibits pancreatic RINm5F ß-cell death caused by oxidative stress. In this study, we examined effects of Tat-DJ-1 protein on streptozotocin (STZ)-induced diabetic mice. Wild type (WT) Tat-DJ-1 protein transduced into pancreas where it markedly inhibited pancreatic ß-cell destruction and regulated levels of serum parameters including insulin, alkaline phosphatase (ALP), and free fatty acid (FFA) secretion. In addition, transduced WT Tat-DJ-1 protein significantly inhibited the activation of NF-κB and MAPK (ERK and p38) expression as well as expression of COX-2 and iNOS in STZ exposed pancreas. In contrast, treatment with C106A mutant Tat-DJ-1 protein showed no protective effects. Collectively, our results indicate that WT Tat-DJ-1 protein can significantly ameliorate pancreatic tissues in STZ-induced diabetes in mice. [BMB Reports 2018; 51(7): 362-367].

Tat-biliverdin reductase A protects INS-1 cells from human islet amyloid polypeptide-induced cytotoxicity by alleviating oxidative stress and ER stress.

Human islet amyloid polypeptide (hIAPP), a major constituent of islet amyloid deposits, induces pancreatic ß-cell apoptosis and eventually contributes to ß-cell deficit in patients with type 2 diabetes mellitus (T2DM). In this study, Tat-mediated transduction of biliverdin reductase A (BLVRA) was investigated in INS-1 cells to examine whether exogenous supplementation of BLVRA prevented hIAPP-induced apoptosis and dysfunction in insulin secretion in ß-cells. Tat-BLVRA fusion protein was efficiently delivered into INS-1 cells in a time- and dose-dependent manner. Exposure of cells to hIAPP induced apoptotic cell death, which was dose-dependently inhibited by pre-treatment with Tat-BLVRA for 1 h. Transduced Tat-BLVRA reduced hIAPP-evoked generation of reactive oxygen species, a crucial mediator of ß-cell destruction. Immunoblot analysis showed that Tat-BLVRA suppressed hIAPP-induced increase in the levels of proteins involved in endoplasmic reticulum (ER) stress and apoptosis signaling. Transduced Tat-BLVRA also recovered hIAPP-induced dysfunction in basal and glucose-stimulated insulin secretions. These results suggested that transduced Tat-BLVRA enhanced the tolerance of ß-cells against IAPP-induced cytotoxicity by alleviating oxidative stress and ER stress. Therefore, Tat-mediated transduction of BLVRA may provide a potential tool to ameliorate ß-cell deficit in pancreas with T2DM.

Alteration of Lysophosphatidylcholine-Related Metabolic Parameters in the Plasma of Mice with Experimental Sepsis.

Plasma concentration of lysophosphatidylcholine (LPC) was reported to decrease in patients with sepsis. However, the mechanisms of sepsis-induced decrease in plasma LPC levels are not currently well known. In mice subjected to cecal ligation and puncture (CLP), a model of polymicrobial peritoneal sepsis, we examined alterations in LPC-related metabolic parameters in plasma, i.e., the plasma concentration of LPC-related substances (i.e., phosphatidylcholine (PC) and lysophosphatidic acid (LPA)), and activities or levels in the plasma of some enzymes that can be involved in the regulation of plasma LPC concentration (i.e., secretory phospholipase A2 (sPLA2), lecithin:cholesterol acyltransferase (LCAT), acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), and autotaxin (ATX)), as well as plasma albumin concentration. We found that levels of LPC and albumin and enzyme activities of LCAT, ATX, and sPLA2 were decreased, whereas levels of PC, LPA, and LPCAT1-3 were increased in the plasma of mice subjected to CLP. Bacterial peritonitis led to alterations in all the measured LPC-related metabolic parameters in the plasma, which could potentially contribute to sepsis-induced decrease in plasma LPC levels. These findings could lead to the novel biomarkers of sepsis.

Tat-PRAS40 prevent hippocampal HT-22 cell death and oxidative stress induced animal brain ischemic insults.

Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and is known to play an important role against reactive oxygen species-induced cell death. However, the precise function of PRAS40 in ischemia remains unclear. Thus, we investigated whether Tat-PRAS40, a cell-permeable fusion protein, has a protective function against oxidative stress-induced hippocampal neuronal (HT-22) cell death in an animal model of ischemia. We showed that Tat-PRAS40 transduced into HT-22 cells, and significantly protected against cell death by reducing the levels of H2O2 and derived reactive species, and DNA fragmentation as well as via the regulation of Bcl-2, Bax, and caspase 3 expression levels in H2O2 treated cells. Also, we showed that transduced Tat-PARS40 protein markedly increased phosphorylated RRAS40 expression levels and 14-3-3σ complex via the Akt signaling pathway. In an animal ischemia model, Tat-PRAS40 effectively transduced into the hippocampus in animal brain and significantly protected against neuronal cell death in the CA1 region. We showed that Tat-PRAS40 protein effectively transduced into hippocampal neuronal cells and markedly protected against neuronal cell damage. Therefore, we suggest that Tat-PRAS40 protein may be used as a therapeutic protein for ischemia and oxidative stress-induced brain disorders.

Tat-ATOX1 inhibits streptozotocin-induced cell death in pancreatic RINm5F cells and attenuates diabetes in a mouse model.

Antioxidant 1 (ATOX1) functions as an antioxidant against hydrogen peroxide and superoxide, and therefore may play a significant role in many human diseases, including diabetes mellitus (DM). In the present study, we examined the protective effects of Tat-ATOX1 protein on streptozotocin (STZ)-exposed pancreatic insulinoma cells (RINm5F) and in a mouse model of STZ-induced diabetes using western blot analysis, immunofluorescence staining and MTT assay, as well as histological and biochemical analysis. Purified Tat-ATOX1 protein was efficiently transduced into RINm5F cells in a dose- and time-dependent manner. Additionally, Tat-ATOX1 protein markedly inhibited reactive oxygen species (ROS) production, DNA damage and the activation of Akt and mitogen activated protein kinases (MAPKs) in STZ-exposed RINm5F cells. In addition, Tat-ATOX1 protein transduced into mice pancreatic tissues and significantly decreased blood glucose and hemoglobin A1c (HbA1c) levels as well as the body weight changes in a model of STZ-induced diabetes. These results indicate that transduced Tat-ATOX1 protein protects pancreatic ß-cells by inhibiting STZ-induced cellular toxicity in vitro and in vivo. Based on these findings, we suggest that Tat-ATOX1 protein has potential applications as a therapeutic agent for oxidative stress-induced diseases including DM.

2,3-Dimethoxy-2'-hydroxychalcone ameliorates TNF-α-induced ICAM-1 expression and subsequent monocyte adhesiveness via NF-kappaB inhibition and HO-1 induction in HaCaT cells.

Up-regulation of adhesion molecules plays an important role in the infiltration of leukocytes into the skin during the development of various inflammatory skin diseases, such as atopic dermatitis. In this study, we investigated the modulatory effects of 2,3-dimethoxy-2'-hydroxychalcone (DMHC) on tumor necrosis factor (TNF)-α-induced intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesiveness, as well as the molecular mechanisms underlying its action in the HaCaT human keratinocyte cell line. Pre-treating HaCaT cells with DMHC significantly suppressed TNF-α-induced ICAM-1 expression and subsequent monocyte adhesiveness. DMHC inhibited TNF-α-induced activation of NF-κB. In addition, DMHC induced HO-1 expression as well as NRF2 activation. Furthermore, HO-1 knockdown using siRNA reversed the inhibitory effect of DMHC on TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that DMHC may inhibit TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes by suppressing the signaling cascades leading to NF-κB activation and inducing HO-1 expression in keratinocytes.

Transduction of PEP-1-heme oxygenase-1 into insulin-producing INS-1 cells protects them against cytokine-induced cell death.

Pro-inflammatory cytokines play a crucial role in the destruction of pancreatic ß-cells, thereby triggering the development of autoimmune diabetes mellitus. We recently developed a cell-permeable fusion protein, PEP-1-heme oxygenase-1 (PEP-1-HO-1) and investigated the anti-inflammatory effects in macrophage cells. In this study, we transduced PEP-1-HO-1 into INS-1 insulinoma cells and examined its protective effect against cytokine-induced cell death. PEP-1-HO-1 was successfully delivered into INS-1 cells in time- and dose-dependent manner and was maintained within the cells for at least 48 h. Pre-treatment with PEP-1-HO-1 increased the survival of INS-1 cells exposed to cytokine mixture (IL-1ß, IFN-γ, and TNF-α) in a dose-dependent manner. PEP-1-HO-1 markedly decreased cytokine-induced production of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA). These protective effects of PEP-1-HO-1 against cytokines were correlated with the changes in the levels of signaling mediators of inflammation (iNOS and COX-2) and cell apoptosis/survival (Bcl-2, Bax, caspase-3, PARP, JNK, and Akt). These results showed that the transduced PEP-1-HO-1 efficiently prevented cytokine-induced cell death of INS-1 cells by alleviating oxidative/nitrosative stresses and inflammation. Further, these results suggested that PEP-1-mediated HO-1 transduction may be a potential therapeutic strategy to prevent ß-cell destruction in patients with autoimmune diabetes mellitus.

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP+) induced SH-SY5Y neuronal cell death and in a 1-methyl- 4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP+-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases.

The effects of PEP-1-FK506BP on dry eye disease in a rat model.

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases.

Down-regulation of MAPK/NF-κB signaling underlies anti-inflammatory response induced by transduced PEP-1-Prx2 proteins in LPS-induced Raw 264.7 and TPA-induced mouse ear edema model.

Excessive reactive oxygen species (ROS) production plays a crucial role in causing various diseases, including inflammatory disorders. The activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling is implicated in stimulating inflammatory response and cytokines. Peroxiredoxin 2 (Prx2) is a 2-cysteine (Cys) peroxiredoxin capable of removing endogenous hydrogen peroxide (H2O2). PEP-1 peptide, a protein transduction domain, consists of three domains which are used to transduce exogenous therapeutic proteins into cells. The correlation between effectively transduced PEP-1-Prx2 and ROS-mediated inflammatory response is not clear. In the present study, we investigated the protective effects of cell permeable PEP-1-Prx2 on oxidative stress-induced inflammatory activity in Raw 264.7 cells and in a mouse ear edema model after exposure to lipopolysaccharides (LPS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Transduced PEP-1-Prx2 suppressed intracellular ROS accumulation and inhibited the activity of MAPKs and NF-κB signaling that led to the suppression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and cytokines in LPS-induced Raw 264.7 cells and TPA-induced mouse ear edema model. Given these results, we propose that PEP-1-Prx2 has therapeutic potential in the prevention of inflammatory disorders.

The Stimulatory Effect of Essential Fatty Acids on Glucose Uptake Involves Both Akt and AMPK Activation in C2C12 Skeletal Muscle Cells.

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and α-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCθ and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 µM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-ß-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.

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

Fibrillar amyloid-beta (fAß) peptide causes neuronal cell death, which is known as Alzheimer's 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 fAß. We observed that fAß 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 fAß1-42 and fAß25-35 induced superoxide production in BV2 cells. fAß25-35 produced superoxide, although fAß25-35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAß does not seem to be dependent on phagocytosis of fAß. Herein we studied how fAß 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 fAß. Furthermore, fAß 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 fAß. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAß through phosphorylation of p47(PHOX) in microglial BV2 cells.

Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms.

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial ß-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein.

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPAinduced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin- 1 beta (IL-1 ß), IL-6, and tumor necrosis factor-alpha (TNF-α). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-κ B) and phosphorylation of p38 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-κ B and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.

ß-arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser(83) 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 P2Y(12) 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 Tyr(31) and Ser(83) . Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser(83) 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 ß-Arrestin 2 (ß-Arr2) with short hairpin RNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser(83) , indicating that ß-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and ß-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 Tyr(31) by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser(83) 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.

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-1ß, 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.