Seung-Wook Ryu

Modification of Daxx by small ubiquitin-related modifier-1

Small ubiquitin-related modifier-1 (SUMO-1) is a protein that is covalently modified to various cellular proteins and protects cells against both anti-Fas and TNF-induced cell death. Previously, we reported that the C-terminus of Daxx interacted with Ubc9, an E2 type SUMO-1 conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130kDa Daxx appeared and the 130kDa band bound to both anti-HA and anti-FLAG antibodies. This means that Daxx can be covalently modified by SUMO-1. Substitution of K630 and K631 abrogated the modification of Daxx by SUMO-1, implying that K630 and K631 were essential for sumoylation. Daxx (K630, 631A) and Daxx (K634, 636, 637A) in which the putative C-terminal nuclear localization signals (NLSs) were disrupted appeared in the nucleus, suggesting that the C-terminal NLS was not functional. Daxx (K630, 631A), the sumoylation defective mutant, was able to interact with PML and co-localized with PML in the PML oncogenic domains (PODs). Thus, our data show that sumoylation status of Daxx does not affect its presence in PODs.

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin.

Inositol 1,4,5‐trisphosphate (Ins‐1,4,5‐P3) is an important second‐messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+ ‐mobilizing agonists. The fate of Ins‐1,4,5‐P3 is determined by two enzymes, a 3‐kinase and a 5‐phosphomonoesterase. The first enzyme converts Ins‐1,4,5‐P3 to Ins‐1,3,4,5‐P4, whereas the latter forms Ins‐1,4‐P2. Recent studies suggest that Ins‐1,3,4,5‐P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3‐kinase [ ~ 400‐fold purified, specific activity = 0.12 μmol/(min · mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3‐kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins‐1,4,5‐P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins‐1,4,5‐P3, an intracellular Ca2+ mobilizer, to Ins‐1,3,4,5‐P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3‐kinase was developed that is based on the separation of [3‐32 P]Ins‐1,3,4,5‐P4 and [γ‐32P]ATP by thin‐layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 μm, Km for Ins‐1,4,5‐P3 = 0.7 μm, Ki for ADP ‐ 12 μm) and divalent cation specificity (Mg2+ > > Mn2+ > Ca2+) for the 3‐kinase. Studies with various inositol polyphosphates indicate that the substrate‐binding site is quite specific to Ins‐1,4,5‐P3. Nevertheless, Ins‐2,4,5‐P3 could be phosphorylated at a velocity approximately 1/20‐1/30 that of Ins‐1,4,5‐P3.—Ryu, S. H.; Lee, S. Y.; Lee, K.‐Y.; Rhee, S. G. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin. FASEB J. 1: 388‐393; 1987.

Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB†

Hepatitis C virus (HCV) infection results in liver injury and long‐term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor‐alpha (TNF‐α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF‐α induces cell death that can be ameliorated by nuclear factor kappaB (NF‐κB) activation. We investigated the regulation of TNF‐α signal transduction in HCV‐infected cells and identified HCV proteins responsible for sensitization to TNF‐α‐induced cell death. We studied the effect of HCV infection on TNF‐α signal transduction using an in vitro HCV infection model (JFH‐1, genotype 2a) with Huh‐7 and Huh‐7.5 cells. We found that TNF‐α‐induced cell death significantly increased in HCV‐infected cells. HCV infection diminished TNF‐α‐induced phosphorylation of IκB kinase (IKK) and inhibitor of NF‐κB (IκB), which are upstream regulators of NF‐κB activation. HCV infection also inhibited nuclear translocation of NF‐κB and expression of NF‐κB‐dependent anti‐apoptotic proteins, such as B‐cell lymphoma—extra large (Bcl‐xL), X‐linked inhibitor of apoptosis protein (XIAP), and the long form of cellular‐FLICE inhibitory protein (c‐FLIP). Decreased levels of Bcl‐xL, XIAP, and c‐FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF‐α‐induced NF‐κB activation and enhanced TNF‐α‐induced cell death. Conclusion: HCV infection enhances TNF‐α‐induced cell death by suppressing NF‐κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune‐mediated liver injury in HCV infection. (HEPATOLOGY 2012;56:831–840)

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.

Dynamic fluorescence imaging for multiparametric measurement of tumor vasculature

Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular parameters including vascular density, perfusion rate, and permeability using spatiotemporal profiles of indocyanine green. Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal tissues with high spatial resolution. The multimodality of this method allowed us to find negative spatial correlations between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic drug. We suggest that DyFI could be a useful tool for the preclinical development of anti-angiogenic drugs.

Blockade of VEGF-A suppresses tumor growth via inhibition of autocrine signaling through FAK and AKT

Blockade of VEGF signaling using RNA interferences, a neutralizing antibody, an antagonizing soluble VEGF receptor, and a receptor tyrosine kinase inhibitor induced anti-tumor effects in human astrocytoma U251-MG and fibrosarcoma HT-1080 in vitro in a dose-dependent manner. Furthermore, blockade of VEGF-A using the doxycycline-inducible VEGF-A RNA interference system showed a significant anti-tumor effect in a murine HT-1080-xenograft model. Anti-tumor effect through the blockade of VEGF signaling was mediated by FAK and AKT pathway in vitro and in vivo. These results collectively indicate that VEGF-A and its receptors can act as key inducer of tumor growth as well as angiogenesis.

RG-II from Panax ginseng C.A. Meyer suppresses asthmatic reaction

In asthma, T helper 2 (T(H)2)-type cytokines such as interleukin (IL)-4, IL-5, and IL-13 are produced by activated CD4(+) T cells. Dendritic cells played an important role in determining the fate of naive T cells into either T(H)1 or T(H)2 cells. We determined whether RG-II regulates the T(H)1/T(H)2 immune response by using an ovalbumin-induced murine model of asthma. RG-II reduced IL-4 production but increased interferon- gamma production, and inhibited GATA-3 gene expression. RG-II also inhibited asthmatic reactions including an increase in the number of eosinophils in bronchoalveolar lavage fluid, an increase in inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyperresponsiveness. This study provides evidence that RG-II plays a critical role in ameliorating the pathogenic process of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of RG-II in terms of its effects in a murine model of asthma.

Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like Cell Death via Intrinsic Reactive Oxygen Cascades

High-intensity femtosecond lasers have recently been used to irreversibly disrupt nanoscale structures, such as intracellular organelles, and to modify biological functions in a reversible manner: so-called nanosurgery and biophotomodulation. Femtosecond laser pulses above the threshold intensity sufficient for reversible biophotomodulation can cause irreversible changes in the irradiated cell, eventually leading to cell death. Here, we demonstrated that cytosolic irradiation with a femtosecond laser produced intrinsic cascades of reactive oxygen species (ROS), which led to rapid apoptosis-like cell death via a caspase and poly (ADP-ribose) polymerase 1 (PARP-1) signaling pathway. We further showed that cells with enhanced mitochondrial fusion activity are more resilient to laser-induced stress compared to those with enforced mitochondrial fission. Taken together, these findings provide fundamental insight into how optical stimulation intervenes in intrinsic cellular signaling pathways and functions.

Tumor-conditioned Gr-1+CD11b+ myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro

Gr-1(+)CD11b(+) cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1(+)CD11(+) cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1(+)CD11b(+) cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1(+)CD11b(+) cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1(+)CD11b(+) cells. Incubation of Gr-1(+)CD11b(+) cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1(+)CD11b(+) cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1(+)CD11b(+) myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1(+)CD11b(+) cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.

Downregulation of OPA3 Is Responsible for Transforming Growth Factor-β-Induced Mitochondrial Elongation and F-Actin Rearrangement in Retinal Pigment Epithelial ARPE-19 Cells

Transforming growth factor-β signaling is known to be a key signaling pathway in the induction of epithelial–mesenchymal transition. However, the mechanism of TGF-β signaling in the modulation of EMT remains unclear. In this study, we found that TGF-β treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-β treatment. Suppression of TGF-β signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-β. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-β-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-β-induced EMT.