Peter Chang-Whan Lee

Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response

The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5WT in PDCD5−/− MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

Impairment of social behavior and communication in mice lacking the Uba6-dependent ubiquitin activation system.

The Uba6-Use1 ubiquitin enzyme cascade is a poorly understood arm of the ubiquitin-proteasome system required for mouse development. Recently, we reported that Uba6 brain-specific knockout (termed NKO) mice display abnormal social behavior and neuronal development due to a decreased spine density and accumulation of Ube3a and Shank3. To better characterize a potential role for NKO mice in autism spectrum disorders (ASDs), we performed a comprehensive behavioral characterization of the social behavior and communication of NKO mice. Our behavioral results confirmed that NKO mice display social impairments, as indicated by fewer vocalizations and decreased social interaction. We conclude that UBA6 NKO mice represent a novel ASD mouse model of anti-social and less verbal behavioral symptoms.

Characterization, Gene Cloning, and Sequencing of a Fungal Phytase, PhyA, From Penicillium oxalicum PJ3

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca2+, Cu2+, Zn2+, and phenylmethylsulfonyl fluoride (PMSF). The Km value for sodium phytate was 0.545 mM with a Vmax of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.

Rehmannia glutinosa polysaccharide induces toll-like receptor 4 dependent spleen dendritic cell maturation and anti-cancer immunity

ABSTRACT Rehmannia glutinosa polysaccharide (RGP) has shown an activation of immune cells in vitro. However, the immune stimulatory effect of RGP in a mouse in vivo is not well studied. In this study, we examined the effect of RGP on dendritic cell (DC) activation and anticancer immunity in vivo. Treatments of RGP in C56BL/6 mice induced increased levels of co-stimulatory molecule expression and pro-inflammatory cytokine production in spleen DCs dependent on toll-like receptor 4 (TLR4), and those DCs promoted interferon-gamma (IFNγ) production in CD4+ and CD8+ T cells. RGP also enhanced ovalbumin (OVA) antigen (Ag)-specific immune activation in tumor-bearing mice, including Ag presentation in DCs, OT-I and OT-II T-cell proliferation, migration of OT-I and OT-II T cells into the B16-OVA tumor, OVA-specific IFNγ production, and the specific killing of OVA-coated splenocytes, which consequently inhibited B16-OVA tumor growth dependent on TLR4 and CD8+ T cells. Finally, the combination of RGP and self-Ag treatment efficiently inhibited CT26 carcinoma and B16 melanoma tumor growth in BLAB/c and C57BL/6 mice, respectively. These data demonstrate that RGP could be a useful adjuvant molecule for immunotherapy against cancer.

Time-dependent effect of E. coli LPS in spleen DC activation in vivo: Alteration of numbers, expression of co-stimulatory molecules, production of pro-inflammatory cytokines, and presentation of antigens.

&NA; Lipopolysaccharide (LPS) is a well‐known stimuli of dendritic cells (DCs). However, in vivo spleen DC maturation by Escherichia coli (E.coli) LPS has not been fully investigated. In this study, we examined the effect of LPS on the activation of spleen DCs and its subsets in a time‐dependent manner on mice in vivo. The frequency, number and migration of spleen conventional DCs (cDCs) were increased 6 and 12 h after completion of LPS treatment. Those increased DC numbers in spleen were then gradually decreased with apoptosis of the DCs. The highest levels of co‐stimulatory molecule expression in the spleen cDCs and their subsets occurred 18 h after LPS treatment, while the pro‐inflammatory cytokines reached their maximum in the intracellular levels of the spleen cDCs and their subsets 3 h after LPS treatment. The antigen presentation of the spleen cDCs and their subsets increased gradually from 3 to 12 h after LPS treatment, but those levels decreased rapidly after 18 h post‐LPS treatment. Thus, by highlighting the importance of time in the stimulation of spleen DCs by LPS in mice in vivo, our data provided a model that could be used by immunologists when considering the manipulation of DC functions in vivo for experimental and clinical applications. HighlightsLPS‐induced activation parameters of spleen cDCs occurred different time point.The cDC numbers in spleen were increased 6 and 12 h after LPS treatment, and the numbers were gradually decreased with apoptosis.Maximum levels of co‐stimulatory molecule expression in the spleen DCs were 18 hours after LPS treatment.The intracellular IL‐6, IL‐12, and TNF‐&agr; production levels reached their maximum 3 hours after post‐LPS treatment in the spleen DCs.Maximum levels of antigen presentation on the spleen cDCs were 6 to 12 hours after LPS injection, and the levels were rapidly decreased.

Purification, sequencing and evaluation of a divergent phytase from Penicillium oxalicum KCTC6440

A fungal strain producing high levels of phytase was purified to homogeneity from Penicillium oxalicum KCTC6440 (PhyA). The molecular mass of the purified PhyA was 65 kDa and optimal activity occurred at 55°C. The enzyme was stable in a pH range of 4.5-6.5, with an optimum performance at pH 5.5. The Km value for the substrate sodium phytate was 0.48 mM with a Vmax of 672 U/mg. The enzyme was inhibited by Ca(2+), Cu(2+), and Zn(2+), and slightly enhanced by EDTA. The PhyA efficiently released phosphate from feedstuffs such as soybean, rich bran and corn meal. The PhyA gene was cloned in two steps of degenerate PCR and inverse PCR and found to comprise 1501 bp and encode 461 amino acid residues. The enzyme was found to have only 13 amino acids differing to the known PhyA from other Penicillium sp., but has distinct enzyme characteristics. Computational analysis showed that PhyA possessed more positively charged residues in the active sites compared to other PhyA molecules, which may explain the broader pH spectrum.

Rehmannia glutinosa polysaccharide promoted activation of human dendritic cells

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced maturation of dendritic cells (DCs) and that it had an anticancer effect in mice. The effect of RGP has not been studied in human DCs, including monocyte-derived DCs (MDDCs) and peripheral blood DCs (PBDCs). In this study, we examined DC activation by RGP in human cells. The dendritic morphology of RGP-treated MDDCs was substantially altered as compared with that of phosphate-buffered saline (PBS)-treated control cells. Moreover, RGP treatment markedly decreased phagocytic activity and increased expression levels of co-stimulatory molecules in MDDCs. In addition, RGP treatment elevated the production of proinflammatory cytokines. Furthermore, RGP-induced activation of MDDCs was dependent on the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RGP-treated MDDCs promoted upregulation of T-cell activation, including proliferation and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production. Analysis of the effect of RGP in PBDC subsets revealed that it induced upregulation of co-stimulatory molecule expression and proinflammatory cytokine production. These data suggest that RGP may function as an immune stimulatory molecule in humans.

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Rehmannia glutinosa polysaccharide functions as a mucosal adjuvant to induce dendritic cell activation in mediastinal lymph node

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced activation of dendritic cells (DCs) in human and mouse subjects. In this study, we evaluated the effect of RGP as a mucosal adjuvant for inducting activation of DCs in the mediastinal lymph node (mLN) in the mouse. The C57BL/6 mice were intranasally (i.n.) treated with RGP and activation of DC in the mLN was analyzed. The treatment with RGP induced a substantial increase in the number of DCs in the mLN due to the up-regulation of C-C motif chemokine receptor 7 (CCR7) in the DCs. Moreover, the expression of co-stimulatory molecules in the mLN DCs and the concentration of pro-inflammatory cytokines in the lung were up-regulated by RGP treatment. Also, RGP treatment induced interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production in the mLN T cells. The combination treatment of RGP and ovalbumin (OVA) induced OVA-specific TCR transgenic I (OT-I) and OT-II cell proliferation in the mLN. Finally, the combination treatment of RGP and tyrosinase-related protein 2 (TRP2) peptide, a melanoma self-antigen, protected mice from melanoma challenge. Thus, these data demonstrated that RGP can be used as a mucosal adjuvant for inducing activation of immune responses in the lung.

Corrigendum: Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response.

Nature Communications 6: Article number: 7390 (2015); 10.1038/ncomms8390 Published: June162015; Updated August212015.