Sang-Muk Oh

Effect of wild ginseng on scopolamine‐induced acetylcholine depletion in the rat hippocampus

Objectives The ameliorating effects of wild ginseng on learning and memory deficits were investigated in rats.

Withaferin A Inhibits Helicobacter pylori-induced Production of IL-1β in Dendritic Cells by Regulating NF-κB and NLRP3 Inflammasome Activation

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. There is evidence that IL-1β is associated with the development of gastric cancer. Therefore, downregulation of H. pylori-mediated IL-1β production may be a way to prevent gastric cancer. Withaferin A (WA), a withanolide purified from Withania somnifera, is known to exert anti-inflammatory and anti-tumor effects. In the present study, we explored the inhibitory activity of WA on H. pylori-induced production of IL-1β in murine bone marrow-derived dendritic cells (BMDCs) and the underlying cellular mechanism. Co-treatment with WA decreased IL-1β production by H. pylori in BMDCs in a dose-dependent manner. H. pylori-induced gene expression of IL-1β and NLRP3 (NOD-like receptor family, pyrin domain containing 3) were also suppressed by WA treatment. Moreover, IκB-α phosphorylation by H. pylori infection was suppressed by WA in BMDCs. Western blot analysis revealed that H. pylori induced cleavage of caspase-1 and IL-1β, as well as increased procaspase-1 and pro IL-1β protein levels, and that both were suppressed by co-treatment with WA. Finally, we determined whether WA can directly inhibit ac tivation of the NLRP3 inflammasome. NLRP3 activators induced IL-1β secretion in LPS-primed macrophages, which was inhibited by WA in a dose-dependent manner, whereas IL-6 production was not affected by WA. Moreover, cleavage of IL-1β and caspase-1 by NLRP3 activators was also dose-dependently inhibited by WA. These findings suggest that WA can inhibit IL-1β production by H. pylori in dendritic cells and can be used as a new preventive and therapeutic agent for gastric cancer.

Nucleotide-binding oligomerization domain 2 (Nod2) is dispensable for the innate immune responses of macrophages against Yersinia enterocolitica

Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for muramyl dipeptide, a component of bacterial peptidoglycan. In this study, we have examined whether Nod2 mediates the immune response of macrophages against Yersinia enterocolitica. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Nod2-deficient mice and were infected with various strains of Y. enterocolitica. ELISA showed that the production of IL-6 and TNF-α in BMDMs infected with Y. enterocolitica was not affected by the Nod2 deficiency. iNOS mRNA expression was induced in both WT and Nod2-deficienct BMDMs in response to Y. enterocolitica, beginning 2 h after infection. Nitric oxide (NO) production by Y. enterocolitica did not differ between WT and Nod2-deficient BMDMs. Western blot analysis revealed that Y. enterocolitica induces activation of NF-κB, p38, and ERK MAPK through a Nod2-independent pathway. Neither LDH release by Y. enterocolitica nor the phagocytic activity of the macrophages was altered by Nod2 deficiency. An in vivo experiment showed that bacterial clearance ability and production of IL-6 and KC in serum were comparable in WT and Nod2-deficient mice infected with Y. enterocolitica. These findings suggest that Nod2 may not be critical for initiating the innate immune response of macrophages against Yersinia infection.

RIP2/RICK‐Dependent Cytokine Production Upon Yersinia enterocolitica Infection in Macrophages with TLR4 Deficiency

Receptor‐interacting protein 2 (RIP2) is a caspase recruitment domain (CARD)‐containing serine/threonine kinase that is activated by NOD1 or NOD2 recognition of their ligands and essential for the activation of NF‐κB and mitogen‐activated protein kinase (MAPK). RIP2 has been known to play an important role in innate immune responses against certain bacterial infection. However, the role and interplay of RIP2 with TLR signalling on cytokine production in macrophages against Yersinia enterocolitica infection remains poorly understood. In the present study, we examined whether RIP2 is essential for Yersinia‐induced production of cytokines in macrophages. Our results showed that naïve RIP2‐deficient macrophages produced similar level of IL‐6, TNF‐α and IL‐10 upon Y. enterocolitica infection compared with wild‐type macrophages. However, the production of IL‐6, TNF‐α and IL‐10 by Y. enterocolitica was impaired in RIP2‐deficient macrophages after lipopolysaccharide (LPS) pretreatment, a TLR4‐tolerant condition. In addition, RIP2 inhibitors, SB203580, PP2, and gefitinib, reduced IL‐6 production in TLR4‐deficient macrophages in response to Y. enterocolitica, whereas they did not affect the cytokines production in WT cells. These results demonstrate that RIP2 may play an important role in proinflammatory cytokine production in macrophages at the absence of TLR signalling.

Phosphorylation of IκBα at serine 32 by T-lymphokine-activated killer cell-originated protein kinase is essential for chemoresistance against doxorubicin in cervical cancer cells.

Background: The regulatory mechanism of TOPK underlying cancer cell survival remains unknown. Results: TOPK directly interacts with and phosphorylates IκBα at Ser-32. Conclusion: TOPK is a critical mediator of cervical cancer chemoresistance in response to doxorubicin. Significance: This study provides new insight on role of TOPK in cervical cancer cell survival in response to doxorubicin. T-lymphokine-activated killer cell-originated protein kinase (TOPK) is known to be up-regulated in cancer cells and appears to contribute to cancer cell proliferation and survival. However, the molecular mechanism by which TOPK regulates cancer cell survival still remains elusive. Here we show that TOPK directly interacted with and phosphorylated IκBα at Ser-32, leading to p65 nuclear translocation and NF-κB activation. We also revealed that doxorubicin promoted the interaction between nonphosphorylated or phosphorylated TOPK and IκBα and that TOPK-mediated IκBα phosphorylation was enhanced in response to doxorubicin. Also, exogenously overexpressed TOPK augmented transcriptional activity driven by either NF-κB or inhibitor of apoptosis protein 2 (cIAP2) promoters. On the other hand, NF-κB activity including IκBα phosphorylation and p65 nuclear translocation, as well as cIAP2 gene expression, was markedly diminished in TOPK knockdown HeLa cervical cancer cells. Moreover, doxorubicin-mediated apoptosis was noticeably increased in TOPK knockdown HeLa cells, compared with control cells, which resulted from caspase-dependent signaling pathways. These results demonstrate that TOPK is a molecular target of doxorubicin and mediates doxorubicin chemoresistance of HeLa cells, suggesting a novel mechanism for TOPK barrier of doxorubicin-mediated cervical cancer cell apoptosis.

Suppression of NF-κB by dieckol extracted from Ecklonia cava negatively regulates LPS induction of inducible nitric oxide synthase gene.

Dieckol, extracted from brown algae, Ecklonia cava, is suggested to elicit anti-inflammatory or anti-tumorigenic activities. However, dieckol-mediated regulatory mechanism for inflammatory response still remains elusive. Here, we show that dieckol suppressed lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression in mouse leukemic macrophage Raw264.7 cells. Also, dieckol decreased LPS-induced both nitric oxide (NO) production and iNOS promoter-driven transcriptional activity in a dose-dependent manner. On the other hand, LPS-mediated NF-κB activity was inhibited by dieckol treatment. Moreover, results revealed that dieckol diminished LPS-mediated p65 nuclear translocation or IκBα phosphorylation dose-dependently, and reduced LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), significantly p38MAPK. Collectively, these findings suggest that dieckol acts as a negative regulator of LPS-mediated iNOS induction through suppression of NF-κB activity, implying a mechanistic role of dieckol in regulation of inflammatory response.

Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF) mediates innate immune responses in murine peritoneal mesothelial cells through TLR3 and TLR4 stimulation.

Abstract Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF), one of the two major TLRs–adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-β in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

Pc2-mediated SUMOylation of WWOX is essential for its suppression of DU145 prostate tumorigenesis

Tumor suppressor WW domain‐containing oxidoreductase (WWOX) is depleted in various cancer types. Here we report that WWOX is modified by small ubiquitin‐like modifier (SUMO) proteins and represses DU145 prostate cancer tumorigenesis in a SUMOylation‐dependent manner. Ectopic WWOX was shown to associate with SUMO2/3 or E2 Ubc9. Furthermore, we revealed that WWOX SUMOylation was promoted by E3 ligase polycomb2 (Pc2), and that WWOX associated with Pc2. Meanwhile, anisomycin‐induced activator protein‐1 (AP‐1) activity was markedly diminished by co‐expression of SUMO and WWOX. Also, WWOX wild type (WT), but not WWOX SUMO mutant (K176A) markedly reduced both DU145 prostate cancer cell proliferation and xenograft tumorigenesis. Collectively, our findings demonstrate that SUMO modification of WWOX is essential for its suppressive activity for DU145 prostate cancer tumorigenesis.

A role for Toll-like receptor 4 in the host response to the lung infection of Yersinia pseudotuberculosis in mice.

Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological features induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain, IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we generated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen, liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient mice. In addition, the levels of TNF-α and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of Yptb infection for mimicking Y. pestis infection.

SUMO Proteins are not Involved in TGF-β1-induced, Smad3/4-mediated Germline α Transcription, but PIASy Suppresses it in CH12F3-2A B Cells

TGF-β induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-β signal-transducing transcription factors, mediate germline (GL) α transcription induced by TGF-β1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-β-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-β1-induced, Smad3/4-mediated GLα transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-β1-induced, Smad3/4-mediated GLα promoter activity, expression of endogenous GLα transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-β1-induced, Smad3/4-mediated GLα promoter activity. We found that PIASy overexpression suppresses the GLα promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLα transcription and IgA switching induced by TGF-β1/Smad3/4, while PIASy acts as a repressor.