Hyeon Ji Yeo

PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1-SIRT2 protein. Purified PEP-1-SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H₂O₂)-induced cell death and cytotoxicity. Also, transduced PEP-1-SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1-SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1-SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1-SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1-SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.

Transduced PEP-1-PON1 proteins regulate microglial activation and dopaminergic neuronal death in a Parkinson's disease model.

Parkinsons disease (PD) is an oxidative stress-mediated neurodegenerative disorder caused by selective dopaminergic neuronal death in the midbrain substantia nigra. Paraoxonase 1 (PON1) is a potent inhibitor of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) against oxidation by destroying biologically active phospholipids with potential protective effects against oxidative stress-induced inflammatory disorders. In a previous study, we constructed protein transduction domain (PTD) fusion PEP-1-PON1 protein to transduce PON1 into cells and tissue. In this study, we examined the role of transduced PEP-1-PON1 protein in repressing oxidative stress-mediated inflammatory response in microglial BV2 cells after exposure to lipopolysaccharide (LPS). Moreover, we identified the functions of transduced PEP-1-PON1 proteins which include, mitigating mitochondrial damage, decreasing reactive oxidative species (ROS) production, matrix metalloproteinase-9 (MMP-9) expression and protecting against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in SH-SY5Y cells. Furthermore, transduced PEP-1-PON1 protein reduced MMP-9 expression and protected against dopaminergic neuronal cell death in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. Taken together, these results suggest a promising therapeutic application of PEP-1-PON1 proteins against PD and other inflammation and oxidative stress-related neuronal diseases.

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult.

BACKGROUND Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1-6) that can reduce hydrogen peroxide (H2O2) and organic hydroperoxides to water and alcohols. METHODS We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis. RESULTS Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H2O2-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H2O2-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage. CONCLUSIONS These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.

Transduced Tat-DJ-1 protein inhibits cytokines-induced pancreatic RINm5F cell death.

Loss of pancreatic β-cells by oxidative stress or cytokines is associated with diabetes mellitus (DM). DJ-1 is known to as a multifunctional protein, which plays an important role in cell survival. We prepared cell permeable wild type (WT) and mutant type (M26I) Tat-DJ-1 proteins to investigate the effects of DJ-1 against combined cytokines (IL-1β, IFN-γ and TNF-α)-induced RINm5F cell death. Both Tat-DJ-1 proteins were transduced into RINm5F cells. WT Tat-DJ-1 proteins significantly protected against cell death from cytokines by reducing intracellular toxicities. Also, WT Tat-DJ-1 proteins markedly regulated cytokines-induced pro- and anti-apoptosis proteins. However, M26I Tat-DJ-1 protein showed relatively low protective effects, as compared to WT Tat-DJ-1 protein. Our experiments demonstrated that WT Tat-DJ-1 protein protects against cytokine-induced RINm5F cell death by suppressing intracellular toxicities and regulating apoptosisrelated protein expression. Thus, WT Tat-DJ-1 protein could potentially serve as a therapeutic agent for DM and cytokine related diseases. [BMB Reports 2016; 49(5): 297-302]

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.

Tat-DJ-1 enhances cell survival by inhibition of oxidative stress, NF-κB and MAPK activation in HepG2 cells

ObjectivesTo identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein.ResultsBy using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H2O2-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H2O2-exposed HepG2 cells.ConclusionsOxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice.

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.

Tat-HSP22 inhibits oxidative stress-induced hippocampal neuronal cell death by regulation of the mitochondrial pathway

Oxidative stress plays an important role in the progression of various neuronal diseases including ischemia. Heat shock protein 22 (HSP22) is known to protect cells against oxidative stress. However, the protective effects and mechanisms of HSP22 in hippocampal neuronal cells under oxidative stress remain unknown. In this study, we determined whether HSP22 protects against hydrogen peroxide (H2O2)-induced oxidative stress in HT-22 using Tat-HSP22 fusion protein. We found that Tat-HSP22 transduced into HT-22 cells and that H2O2-induced cell death, oxidative stress, and DNA damage were significantly reduced by Tat-HSP22. In addition, Tat-HSP22 markedly inhibited H2O2-induced mitochondrial membrane potential, cytochrome c release, cleaved caspase-3, and Bax expression levels, while Bcl-2 expression levels were increased in HT-22 cells. Further, we showed that Tat-HSP22 transduced into animal brain and inhibited cleaved-caspase-3 expression levels as well as significantly inhibited hippocampal neuronal cell death in the CA1 region of animals in the ischemic animal model. In the present study, we demonstrated that transduced Tat-HSP22 attenuates oxidative stress-induced hippocampal neuronal cell death through the mitochondrial signaling pathway and plays a crucial role in inhibiting neuronal cell death, suggesting that Tat-HSP22 protein may be used to prevent oxidative stress-related brain diseases including ischemia.

Cytokine-induced apoptosis inhibitor-1 causes dedifferentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

Cytokine-induced apoptosis inhibitor-1 (CIAPIN-1, formally named anamorsin) is a well-known regulator of apoptosis in many different cell types. Recently, it has been reported that some anti-apoptotic proteins are involved with the regulation of cell differentiation. However, relatively little is known about the role of CIAPIN-1 on rabbit articular chondrocytes differentiation. In this study, we investigated the effects of CIAPIN-1 in chondrocytes, focusing on extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling. CIAPIN-1 caused dedifferentiation, as determined by the inhibition of type II collagen expression and sulfated-proteoglycan synthesis. CIAPIN-1 activated ERK-1/2 and inactivated p38 kinase, as determined by the phosphorylation level of each protein. CIAPIN-1-induced ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which also prevented the CIAPIN-1-induced loss of type II collagen expression. Inhibition of p38 kinase with SB203580 enhanced the decrease in type II collagen expression. Our findings collectively suggest that ERK-1/2 and p38 kinase regulate CIAPIN-1-induced dedifferentiation in rabbit articular chondrocytes.

Tat-DJ-1 inhibits oxidative stress-mediated RINm5F cell death through suppression of NF-κB and MAPK activation

Oxidative stress is highly involved in the development of diabetes mellitus by destruction of pancreatic β-cells. DJ-1 is an antioxidant protein and DJ-1 expression levels are known to be reduced in diabetes mellitus. Thus, we examined the effects of DJ-1 protein against oxidative stress-induced pancreatic β-cell (RINm5F) death using cell permeable wild-type and mutant-type (C106A) Tat-DJ-1 proteins, which both efficiently transduced into RINm5F cells. Intracellular stability of wild-type Tat-DJ-1 persisted two times longer than C106A Tat-DJ-1. Wild-type Tat-DJ-1 protein markedly protected cells from hydrogen peroxide-induced toxicities such as cell death, reactive oxygen species generation, and DNA fragmentation. Further, wild-type Tat-DJ-1 protein significantly inhibited hydrogen peroxide-induced activation of mitogen-activated protein kinases and NF-κB signaling. On the other hand, C106A Tat-DJ-1 protein did not show the same protective effects. These results indicate that wild-type Tat-DJ-1 inhibits oxidative stress-induced cellular toxicity and activation of mitogen-activated protein kinases and NF-κB signals in RINm5F cells. These results suggest that wild-type Tat-DJ-1 protein may be a potential therapeutic agent against diabetes mellitus or toward the prevention of pancreatic β-cell destruction.