Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post- translational modification of IκB

장광,이선진,정일영,Timothy R. Billiar,정헌택,한정아,권영근,하권수,김영명 생화학분자생물학회 2004 Experimental and molecular medicine Vol.36 No.4

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the m olecular m echanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2 -, or NO3 - did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN- γ -stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1β, IL-6 and TNF-α mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl- L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokineinduced increase in NF-κB activation, iNOS promoter activity, nuclear translocation of cytosolic NF-κB p65 subunit, and IκBα degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of IκB. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of IκBα thereby preventing NF-κB activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.

Hypoxia Activates Toll-like Receptor 4 Signaling in Primary Mouse Hepatocytes Through the Receptor Clustering within Lipid Rafts

Dong Hee Kim,Timothy R. Billiar 대한외과학회 2011 Annals of Surgical Treatment and Research(ASRT) Vol.80 No.3

Purpose: Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. Methods: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8∼12 weeks old) by an in situ collagenase (Type Ⅳ, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. Results: Hypoxia caused TLR4/MD2 and β2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. Conclusion: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.

Signaling and Function of Caspase and c-Jun N-terminal Kinase in Cisplatin-induced Apoptosis

Myoung-Sook Koo,권영근,Joon-Hong Park,Won-Jin Choi,TimothyR. Billiar,김영명 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.2

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wildtype and JB-6 cells, and the caspase-3 inhibitor ZDEVD- fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin- induced apoptotic signal is initiated by the caspase- 8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Do, Hang,Kang, Nam‐,Sung,Pyo, Suhkneung,Billiar, Timothy R.,Sohn, Eun‐,Hwa Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of cellular biochemistry Vol.111 No.5

<P><B>Abstract</B></P><P>Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.</P>

Nitric Oxide as a Bioregulator of Apoptosis

Chung, Hun-Taeg,Pae, Hyun-Ock,Choi, Byung-Min,Billiar, Timothy R.,Kim, Young-Myeong 원광대학교 생명공학연구소 2001 생명공학연구소보 Vol.8 No.1

Nitric oxide (NO), synthesized from L-arginine by NO synthases, is a small, diffusible, highly reactive molecule with dichotomous regulatory roles under physiological and pathological conditions. NO can promote apoptosis (proapoptosis) in some cells, whereas it inhibits apoptosis (antiapoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as iron, thiols, proteins, and reactive oxygen species. Long-lasting production of NO acts as a pro-apoptotic modulator by activating caspase family proteases through the release of mitochondrial cytochrome c into the cytosol, upregulation of p53 expression, activation of JNK/SAPK, and altering the expression of apoptosis-associated proteins including Bcl-2 family proteins. However, low or physiological concentrations of NO prevent cells from apoptosis induced by trophic factor withdrawal, Fas, TNFα, and lipoplysaccharide. The anti-apoptotic mechanism can be understood via expression of protective genes such as heat shock proteins. Bcl-2 as well as direct inhibition of the apoptotic caspase family proteases by S-mitrosylation of the cysteine thiol. Our current understanding of the mechanisms by which NO exerts both pro- and anti-apoptotic actions is discreased in this review article.

Nitric Oxie Down-regulates Hepatocyte- Inducible Nitric Oxide Synthase Gene Expression

Taylor, Bradley S.,Kim, Young-Myeong,Wang, Qi,Shapiro, Richard A.,Billiar, Timothy R.,Geller, David A. MediMedia Korea 1998 JAMA-Korea Vol.13 No.1

Dexamethasone protects primary cultured hepatocytes from death receptor-mediated apoptosis by upregulation of cFLIP

Oh, H-Y,Namkoong, S,Lee, S-J,Por, E,Kim, C-K,Billiar, T R,Han, J-A,Ha, K-S,Chung, H-T,Kwon, Y-G,Lee, H,Kim, Y-M Nature Publishing Group 2006 CELL DEATH AND DIFFERENTIATION Vol.13 No.3

Dexamethasone (DEX) pretreatment protected hepatocytes from TNF-α plus actinomycin D (ActD)-induced apoptosis by suppressing caspase-8 activation and the mitochondria-dependent apoptosis pathway. DEX treatment upregulated cellular FLICE inhibitory protein (cFLIP) expression, but did not alter the protein levels of Bcl-2, Bcl-xL, Mcl-1, and cIAP as well as Akt activation. The increased cFLIP mRNA level by DEX was inhibited by ActD, indicating that DEX upregulates cFLIP expression at the transcriptional step. DEX also inhibited Jo2-mediated hepatocyte apoptosis by blocking the formation of the death-inducing signaling complex and caspase-8 activation. Specific downregulation of cFLIP expression using siRNA reversed the antiapoptotic effect of DEX by increasing caspase-8 activation. Moreover, DEX administration into mice increased cFLIP expression in the liver and prevented Jo2-induced hepatic injury by inhibiting caspase-8 and -3 activities. Our results indicate that DEX exerts a protective role in death receptor-induced in vitro and in vivo hepatocyte apoptosis by upregulating cFLIP expression.Cell Death and Differentiation (2006) 13, 512–523. doi:10.1038/sj.cdd.4401771; published online 16 September 2005

Protective effect of p53 in vascular smooth muscle cells against nitric oxide-induced apoptosis is mediated by up-regulation of heme oxygenase-2

( Young Myeong Kim ),( Byung Min Choi ),( Yong Seok Kim ),( Young Guen Kwon ),( Melina R Kibbe ),( Timothy R Billiar ),( Edith Tzeng ) 생화학분자생물학회 2008 BMB Reports Vol.41 No.2

Cancer gene therapy using a novel secretable trimeric TRAIL

Kim, C-Y,Jeong, M,Mushiake, H,Kim, B-M,Kim, W-B,Ko, J P,Kim, M-H,Kim, M,Kim, T-H,Robbins, P D,Billiar, T R,Seol, D-W Nature Publishing Group 2006 Gene therapy Vol.13 No.4

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is a type II transmembrane cytokine molecule. Soluble TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth specifically without damaging normal cells and tissues in vivo. In our previous report, we have demonstrated that an artificial gene encoding the polypeptide composed of the three functional elements (a secretion signal, a trimerization domain and an apoptosis-inducing moiety of TRAIL gene sequence) expresses and secretes highly apoptotic trimeric TRAIL into the culture supernatant. Here, as an approach to TRAIL-based cancer gene therapy, we developed an adenoviral vector delivering the gene that encodes our secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vitro in cancer cell lines such as HeLa, MDA-MB-231, A549, HCT116 and U-87MG. In an animal xenograft tumor model bearing a human glioma cell line U-87MG, intratumoral delivery of Ad-stTRAIL dramatically suppressed tumor growth without showing detectable adverse side effects. Histological analysis revealed that Ad-stTRAIL suppresses tumor growth by inducing apoptotic cell death. Contrary to the known rapid clearance of systemically delivered TRAIL protein from the blood circulation, stTRAIL expressed by Ad-stTRAIL in tumor tissues persisted for more than 4 days. In a comparison of tumor suppressor activity between Ad-stTRAIL and Ad-flTRAIL (delivering the full-length TRAIL gene) after mixing infected cells with uninfected cells and implanting these mixed cells in nude mice, Ad-stTRAIL showed higher tumor suppressor activity than that of Ad-flTRAIL. Our data reveal that a gene therapy using Ad-stTRAIL has a promising potential to treat human cancers including gliomas.Gene Therapy (2006) 13, 330–338. doi:10.1038/sj.gt.3302658; published online 29 September 2005