Abstracts Poster Presentation I

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 Poster Presentation I
Poster Presentation I       ❰❰

P-1                                                                               P-4
Inhibition of Hepatic Cytochrome P450 4A (Cyp4a) Reduces                          Functional Study of Smad7 Using Skin-Specific Transgenic
ER Stress and Improves Insulin Resistance in Type 2 Diabetes                      Mouse Model
Edmond Changkyun Park, Yeonhee Hong, Eun-Young Shin, And Gun-Hwa                  Young-Mi Kim, and Suntaek Hong*
Kim*                                                                              Lab. of Cancer Cell Biology, Lee Gil Ya Cancer and Diabetes Institute, Gachon
Division of Life Science, Korea Basic Science Institute, Daejeon 305-333, Korea   University
*Corresponding author: genekgh@kbsi.re.kr                                         *Corresponding author: sthong@gachon.ac.kr

ER stress signaling is responsible for hepatic insulin resistance and can re-     Smad7 is an inhibitory Smad, which suppresses the TGF-β signaling. It is ex-
sult in type 2 diabetes (T2DM). However, the underpinnings of mechanisms          pressed at low levels in normal epithelial tissues, but is overexpressed in cer-
that regulate ER stress and T2DM are poorly understood. Here we show the          tain cancers. Previous reports showed that Smad7 functions as anti-in-
biochemical and physiological characterizations of Cyp4a as a novel regu-         flammatory mediator of TGF-β to block the inflammatory TNF-α signaling.
lator of ER stress-induced hepatic insulin resistance and apoptosis. We           However, its functions in contact hypersensitivity (CHS) are largely unknown.
found that Cyp4a is ighly up-regulated in db/db diabetic mice liver. Inhibition   Therefore, we examined the role of Smad7 using wild-type or mutant Smad7
of Cyp4a in db/db and high fat diet (HFD) mice using its specific inhibitor       overexpressing transgenic mice in CHS. Ear swelling was induced by chal-
HET0016 improved the diabetic physiological phenomena, such as glucose            lenging with DNFB. Pathological phenotype was significantly increased in
tolerance, insulin secretion and hepatic steatosis. The insulin resistance and    mutant Smad7 mice, which does not inhibit TNF- αsignaling, when compared
apoptosis were also rescued by loss of Cyp4a function. We revealed that the       with control or wild-type Smad7 mice. Next, we checked the expression profil-
amelioration of diabetes was due to the reduction of ER stress which was          ing of chemokines and cytokines using qRT-PCR or cytokine array. Finally,
up-regulated in diabetic mice liver. On the contrary, induction of Cyp4a in       we identified that CCL5/RANTES is an important mediator in DNFB-induced
db/db mice accelerated ER stress, insulin resistance and apoptosis. These         CHS and mutant Smad7 promotes the expression of inflammatory cytokine at
results show the importance of Cyp4a in the remission of diabetes and of ER       transcriptional level. This finding suggests that Smad7 has a novel function
stress-induced insulin resistance and apoptosis and suggest that the reduc-       for anti-inflammatory function of TGF-β signaling in resolution stage and may
tion of Cyp4a activity would be a therapeutic target against T2DM.                be a therapeutic target for inflammatory diseases of skin.

P-2                                                                               P-5
Baicalin Improves Chronic Corticosterone-induced Learning                         The Role of Cpsf-2/Cleavage and Polyadenylation Specificity
and Memory Deficits Via Enhancement of Impaired                                   Factor 2 in C. Elegans Lifespan Regulation
Hippocampal BDNF and CREB Expression in the Rat                                                 1                 1                   1                       1,2,3,
                                                                                  Heehwa Son , Dongyeop Lee , Dae-Eun Jeong , and Seung-Jae Lee                *
           1,               2                1               2               1    1                                        2
Bombi Lee *, Bongjun Sur , Sunghun Kim , Jinhee Park , Mijung Yeom ,               Division of Molecular and Life Science, School of Interdisciplinary Bioscience
             1,2             1,2                      1,2,                                           3
Insop Shim , Hyejung Lee , And Dae-Hyun Hahm *                                    and Bioengineering, World Class University Information Technology Convergence
 Acupuncture and Meridian Science Research Center, College of Oriental            Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk,
Medicine, Kyung Hee University, Seoul, 130-701, Korea, 2The Graduate School of    790-784, South Korea
Basic Science of Oriental Medicine, College of Oriental Medicine, Kyung Hee       *Corresponding author: seungjaelee1@gmail.com
University, Seoul, 130-701, Korea.
                                                                                  The effects of abnormal glucose regulation on age-related diseases including
*Corresponding author: bombi@khu.ac.kr
                                                                                  type 2 diabetes have been extensively studied. However, research on how
The purpose of this study was to examine whether baicalin (BAI) improves          glucose influences aging itself has just begun. Recent reports demonstrated
spatial cognitive impairments induced in rats following the repeated admin-       that glucose-enriched diet shortens the lifespan of the C. elegans. By per-
istration of the exogenous stress hormone corticosterone (CORT). The effect       forming a genome-wide RNAi screen using a glucose-inducible fluorescence
of BAI on the hippocampal expression of BDNF and CREB was also                    reporter, we identified many genes that affected the metabolism and the life-
investigated. For 21 days, male rats received daily doses of BAI (20, 50 and      span of C. elegans. Here, we present our finding regarding the role of cpsf-2,
100 mg/kg, i.p.) 1 h prior to a CORT injection. The daily administration of BAI   a cleavage and polyadenylation specificity factor that regulates 3' end cleav-
improved memory impairment as measured by the passive avoidance test              age during RNA processing, in the lifespan regulation. We initially found that
and reduced the escape latency for finding the platform in the Morris water       cpsf-2 RNAi decreased the glucose-inducible fluorescence reporter. Furthermore,
maze test. Additionally, as assessed by immunohistochemistry and RT-PCR           we showed that knockdown of cpsf-2 significantly increased the lifespan of C.
analysis, the administration of BAI also significantly alleviated memo-           elegans. These data indicate that reduction of cpsf-2 may promote long life-
ry-associated decreases in the expression levels of BDNF and CREB pro-            span by reducing metabolism-related genes that play a role in increasing the
teins and mRNA in the hippocampus. Thus, these findings suggest that BAI          glucose level. Currently, we are determining which aging-regulatory genes in-
might be useful as a therapeutic agent in various neurodegenerative dis-          teract with cpsf-2. Our research may help elucidate novel mechanisms by
eases for the improvement of cognitive function. This likely occurs through       which glucose influences lifespan and metabolism.
the regulation of BDNF and CREB expression (This research was supported
by the National Research Foundation of Korea Grant funded by the Korean
Government (MEST)(2010-0003678).

P-3                                                                               P-6
Regulatory Mechanism of Pellino3 for Inflammation-associated                      A Critical Role of Ceramide-mediated ATF3 on Hepatic Lipid
Carcinogenesis in Knockout Mouse Model                                            Accumulation in Metabolic Disease Models: by Changes of
                                                                                  Caveolin-1/CD36 Composition
Jooyoung Kim, and Suntaek Hong*
Laboratory of Cancer Cell Biology, Lee Gil Ya Cancer and Diabetes Institute,      Keon Jae Park, Ji Yeon Kim, Jeong Eun Kim, Gyu Hee Kim, Eun Ae Jung,
Gachon University                                                                 and Won-Ho Kim*
*Corresponding author: sthong@gachon.ac.kr                                        Division of Metabolic Diseases, Center for Biomedical Sciences, National Institutes
                                                                                  of Health
Toll-like receptors are key regulators of innate immune system as primary         *Corresponding author: jhkwh@daum.net
sensors of conserved microbial structures. The intracellular domain of TLRs
is similar to that of members of the interleukin-1 receptor family, which acti-   In the onset and progression of ASH and NASH, ceramide is significantly in-
vate similar intracellular signaling pathways. Pellino proteins are molecules     creased by alterations of sphingolipid. However, the regulatory mechanisms
that interact with the drosophila IRAK-like molecule Pelle. In vitro studies      by which ceramide drives hepatic steatosis remain poorly understood. Here,
suggest Peli proteins function as E3 ubiquitin ligases that catalyze K63-linked   we examined the role of ATF3 on ceramide-mediated liver steatosis in etha-
ubiquitin chains. Peli proteins physically interact with IRAKs and induce ubiq-   nol-fed mice or HFD-fed rats. Hepatic steatosis increased in both models are
uitination of IRAK1. Recent study showed genetic evidence that Peli1 has a        correlated with the increase of ceramide levels in their serum or liver tissues,
non-redundant role in regulating RIP1 ubiquitination and IKK activation in the    accompanied with the expression of caveolin-1, CD36/FAT, and sphingomye-
TRIF-dependent TLR pathway using Pellino1 deficiency mouse model.                 lin synthase-2, which was determined by TNF-a production and TNFR1- de-
However, it is not clear the physiologic functions of other Pellino family.       pendent pathway. Concomitantly, a stress-inducible ATF3 was significantly
Therefore, we are using Pellino3 deficiency mouse model for identification of     increased in these models and it plays as a potent regulator for de novo ce-
regulatory mechanism and physiologic study. Further analyses of these mol-        ramide synthesis and ceramide-mediated lipid accumulation. Also, ATF3 di-
ecules showing a common function in the TLR/IL-1R signaling pathways              rectly regulates caveolin-1 and CD36/FAT expression, thereby increases ce-
might help delineate their roles in immunity and possible crosstalk between       ramide-mediated lipogenesis and inhibits insulin receptor signaling, which
the inflammation and inflammation induced carcinogenesis.                         were abolished by ATF3 siRNA. Taken together, our studies suggest that ce-
                                                                                  ramide-mediated ATF3 is a critical regulatory pathway in dynamic regulation
                                                                                  of lipid microdomains via induction of caveolin-1 and CD36/FAT.

                                                                                                                         The 24th KSMCB Winter Conference 2013          35
❱❱   Poster Presentation I

     P-7                                                                                       P-10
     Activating Transcription Factor-3 plays as a Novel Potent                                 Difference of Differentiation Capacity Among Osteoblasts of
     Regulator of Renal Fibrosis in Streptozotocin-Injected Rats                               Maxilla and Mandible
     Gyu Hee Kim, Jeong Eun Kim, Do Hee Kim, Ji Yeon Kim, Jeong Suk Kang,                      Hoon Joo Yang, Yun Mi Song, Tae Hyung Cho, Li Yeon Kim, In Sook Kim,
     Keon Jae Park, Eun Ae Jeong, and Won-Ho Kim*                                              and Soon Jung Hwang*
     Division of Metabolic Disease, Center for Biomedical Sciences, National Institute         Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul
     of Health                                                                                 National University
     *Corresponding author: jhkwh@daum.net                                                     *Corresponding author: ishwkim@snu.ac.kr

     Diabetic nephropathy, an end-stage disease of diabetic complication, is charac-           The maxilla and mandible shows different property of bone, however, The ca-
     terized by fibrosis of renal glomerulus and tubulointerstitial region. However, the ex-   pacity of differentiation has not been well-known yet. The purpose of this
     act molecular mechanisms by which diabetes may initiate or exacerbate kidney fail-        study is to verify the site-specific difference of osteoblasts for bone formation.
     ure remain elusive. Here, we investigated the role of ATF3 on the induction of dia-       The osteoblast from mandible and maxilla were harvested from the same in-
     betic nephropathy and molecular mechanisms involved in ATF3-mediated fibrosis.            dividual and cultured. The differentiation capacity and osteoblast differ-
     STZ-injected rats exhibited renal dysfunction, as evidenced by increased glomer-          entiation-relating gene expression were evaluated. The osteoblast from man-
     ulus, thickened basement membrane, and increased mesenterium, which are con-              dible and maxilla was treated with Dex and estrogen, which is related with os-
     sistent with higher levels of albuminuria, blood glucose and serum MCP-1. The in-         teoporosis, to evaluate the differentiation capacity and proliferation. When
     filtration of CD68+ cells was also significantly increased in the glomerulus and tubu-    treated with Dex and estrogen, The proliferation of osteoblasts from maxilla
     lointerstitial region, correlated with the marked accumulation of ECM molecules and       and mandible did not have specific tendency. The osteoblasts from maxilla
     ATF3. In NRK-52E cells, TNF-a-induced fibrosis was determined by ATF3 since the           and mandible showed 7~63% increase in the ALP activity with Dex. When
     increased ECM molecules such as MCP1, FN, collagen IV are abolished by ATF3               treated with estrogen, the osteoblasts from maxilla did not showed increase
     depletion. Also, ATF3 plays as a direct transcriptional activator of MCP-1 gene.          in the differentiation capacity, on the other hand, the osteoblasts from man-
     Collectively, we know that ATF3 may play as an important regulator of renal fibrosis      dible showed 14~22% increase in the differentiation capacity with 100nM
     and is considered as a new aspect of the therapeutic mechanism of DN.                     estrogen. The ALP activity of mandibular osteoblasts were higher than that of
                                                                                               maxillary osteoblasts.

     P-8                                                                                       P-11
     Genome-wide Identification of Auxin Response Factor Gene                                  Androstenedione Induces Cell-cycle Arrest and Apoptosis in
     Family in Brassica Rapa                                                                   Human Endometrial Adenocarcinoma Cells
     Jeong-Hwan Mun1, Hee Chung2, Young-Min Jeong2, Mijin Oh1, Hyun-Ju                         Seung Bin Park, and Myung Suk Han*
     Hwang1, and Hee-Ju Yu2,*                                                                  Department of Obstetrics and Gynecology, Dong-A University, College of Medicine,
      Department of Agricultural Biotechnology, National Academy of Agricultural               Dong-A University, Busan, South Korea
     Science, Rural Development Administration, 150 Suin-ro, Gwonseon-gu, Suwon                *Corresponding author: hnsobgy@dau.ac.kr
     441-707, Korea, Department of Life Sciences, The Catholic University of Korea,
     43 Jibong-ro, Wonmi-gu, Bucheon 420-743, Korea                                            Androstenedione (ASD) is an androgen steroid that is normally synthesized
     *Corresponding author: yuheeju@catholic.ac.kr                                             within men and women and may be metabolized to a more potent androgen
                                                                                               or estrogen hormone. ASD is a precursor of testosterone in males and estra-
     In this study, we identified the auxin response factor (ARF) gene family, which is        diol and estrone in females. Akt/MAPK plays a critical role in cell growth and
     one of the key regulators of auxin-mediated plant growth and development. A total         survival. Recent studies have shown that the effect of Akt/MAPK in prostate
     of 31 ARF genes were identified in the genome. Phylogenetic and evolutionary              cancer cell is mediated through androgen. However, the androgen acting to
     analyses suggest that ARF genes fell into four major classes and were amplified in        cell growth and survival in endometrial cells is currently unclear. We verified
     the B. rapa genome as a result of a recent whole genome triplication after speci-         these results in vitro, using the well-differentiated endometrial adenocarcinoma
     ation from Arabidopsis thaliana. Despite its recent hexaploid ancestry, B. rapa in-       cells (Ishikawa cell), and this cell line has been considered to resemble normal
     cludes a relatively small number of ARF genes compared to the 23 members in A.            endometrial epithelium in terms of cell adhesion molecules and has been
     thaliana, presumably due to a paralog reduction related to repetitive sequence in-        used to study the regulation of these molecules. These results indicate that
     sertion into promoter and non-coding transcribed region of the genes. mRNA se-            ASD interfered with Akt activation and MAPK signals through the increasing
     quencing analyses demonstrated that 27 of the 31 BrARF genes were transcription-          apoptosis signals leading to a controlled caspases, Bax genes and protein
     ally active, and their expression was affected by either auxin treatment or floral de-    expression, and reduced cell proliferation caused the arrest of cells in the
     velopment stage. This study will provide a fundamental basis for the modification         G0/G1 phase of the cell cycle.
     and evolution of the gene family after a polyploidy event, as well as a functional
     study of ARF genes in a polyploidy crop species. This work was supported by
     grants from National Research Foundation of Korea (20110013214).

     P-9                                                                                       P-12
     Gains of Ubiquitylation Sites in Highly Conserved Proteins                                Inhibition of ERK5 Protects Pulmonary Fibrosis Via
     during Human Evolution                                                                    Downregulation of TGF-β1-Smad Signaling
                                                                                                        1                 1                1                1                  2
     Dong Seon Kim, and Yoonsoo Hahn*                                                          Suji Kim , Deahwan Nam , Suhyun Park , Junghwa Han , Jae-Hyang Lim ,
     Department of Life Science, Chung-Ang University, Seoul 156-756                           and Chang-Hoon Woo *
     *Corresponding author: yoonsoo.hahn@gmail.com                                              Department of Pharmacology, Aging-associated Vascular Disease Research
                                                                                               Center, Yeungnam University College of Medicine, 317-1 Daemyung-dong, Daegu,
     Post-translational modification of lysine residues of specific proteins by ubiq-          Korea, 2Department of Microbiology, School of Medicine, Ewha Womens University,
     uitin modulates the degradation, localization, and activity of these target               911-1 Mok6-dong, Yangcheon-gu, Seoul, Korea
     proteins. We analyzed human ubiquitylation site data and multiple alignments              *Corresponding author: changhoon_woo@yu.ac.kr
     of orthologous mammalian proteins, and identified 281 ubiquitylation sites in
     252 proteins that first appeared during human evolution. PML, which is in-                TGF-β1-Smad signaling plays an important role in pulmonary fibrosis.
     volved in neurodevelopment and neurodegeneration, acquired three sites,                   TGF-β1-mediated MAPK family activation affects Smad signaling. ERK5 are
     two of which have been reported to be involved in the degradation of PML.                 also involved in promoting hypertrophic remodeling which lead to fibrotic and
     Thirteen human proteins, including ERCC2 and NBR1, gained human-specif-                   ECM gene expression in cardiomyocytes. However, the role of ERK5 in pul-
     ic ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has                 monary fibrosis remains not known. Herein, we identified whether ERK5 reg-
     a K/Q polymorphism, the derived (major) allele of which confers enhanced                  ulates TGF-β1-induced fibrogenic gene expression in both in vitro and in vivo
     DNA repair capacity and reduced cancer risk compared with the ancestral                   system. Pharmacological inhibitor of MEK5/ERK5, BIX02189 and depletion
     (minor) allele. NBR1 and eight other proteins that are involved in the human              of ERK5 with siRNA inhibited TGF-β1-induced ECM molecules in lung epi-
     autophagy protein interaction network gained a novel ubiquitylation site. The             thelial cells and fibroblasts. Inhibition of ERK5 also blocked TGF-β1 signal to
     gain of novel ubiquitylation sites could be involved in the evolution of protein          smad3 transcriptional activity and PAI-1 promoter activity. TGF-β1-induced
     degradation and other regulatory networks.                                                Smad3 phosphorylation and nuclear translocation was not regulated by
                                                                                               ERK5 signaling. Notably, ERK5 is involved in Smad2 phosphorylation at link-
                                                                                               er region and Smad3 acetylation. In bleomycin-induced lung fibrosis, we
                                                                                               found that BIX02189 treatment improved survival rate and inhibited lung fib-
                                                                                               rosis of mice after bleomycin. Our results demonstrate ERK5 plays a major
                                                                                               role in TGF-β1-induced Smad3 acetylation, PAI-1 transcription, and ECM

36    Korean Society for Molecular and Cellular Biology
Poster Presentation I      ❰❰

P-13                                                                                     P-16
Stabilization of p21 (Cip1/WAF1) Following Tip60 Dependent                               Overexpression of the Arabidopsis Vacuolar
Acetylation Is Required for p21Mediated DNA Damage                                       H+-pyrophosphatase AVP1 Gene in Rice Plants Increases
Response                                                                                 Tolerance to Various Environmental Stresses
Min-Sik Lee1, Jinho Seo1, Do young Choi2, Eun-Woo Lee1, Aram Ko1,                        Ho-Nam Lee1, Woo-Jin Park1, Hyun-Jun Jeon1, Dong-Jin Shin1, Hyeon-Oh
Nam-Chul Ha3, Jong Bok Yoon1, Han-Woong Lee1, Kwang Pyo Kim2, and                        Hwong1, Kyoung-Ae Kim1, Young-Saeng Kim2,3, Il-Sup Kim2,3, and Ho-
Jaewhan Song1,*                                                                          Sung Yoon2,3,*
1                                                                                        1
 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei            Gyeonggu High School, 279 Bonggok-dong Gumi-si Gyeongsangbuk-do,
University, Seoul 120-749, Republic of Korea., 2Institute of Biomedical Science          730-200, Korea, 2Department of Biology, Kyungpook National University, Daegu
and Technology, Department of Molecular Biotechnology, Konkuk University,                702-701, Korea, ABRC, Kyungpook National University, Daegu 702-701, Korea
Seoul, Republic of Korea., 3Department of Manufacturing Pharmacy, College of             *Corresponding author: hyoon@knu.ac.kr
Pharmacy, Pusan National University, Busan, Republic of Korea.
*Corresponding author: jso678@yonsei.ac.kr                                               The Arabidopsis gene AVP1 encodes a vacuolar H -translocating inorganic
                                                                                         pyrophosphatase (EC that functions as an electronic proton pump in
The molecular mechanisms controlling post-translational modifications of p21 have        the vacuolar membrane and affects growth development and stress re-
been pursued assiduously in recent years. Here, utilizing mass-spectrometry analy-       sponses in plants. Incorporation and expression of the AVP1 transgene was
sis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at ami-   confirmed by semi-quantitative RT-PCR and quantitative real-time PCR.
no acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for     Soil-grown transgenic rice plants expressing AVP1 (TRP1 and TRP2)
the first time. Detection of adriamycin-induced p21 acetylation, which disappeared       showed significantly enhanced tolerance to 100 mM NaCl under glasshouse
after Tip60 depletion with concomitant destabilization of p21 and disruption of G1       conditions when compared to wild-type (WT) rice plants. Augmented AVP1
arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA dam-          expression in the transgenic rice plants also affected root development and
age-induced cell cycle regulation. The ability of 2KQ, a mimetic of acetylated p21,      total biomass, and improved ion homeostasis through increased accumu-
to induce cell cycle arrest and senescence was significantly enhanced in p21 null        lation of Na+ ions in whole tissues under high salinity conditions when com-
MEFs compared to those of cells expressing wild-type p21. Together, these ob-            pared to WT rice plants. Furthermore, rice grain yield and biomass of the
servations demonstrate that Tip60-mediated p21 acetylation is a novel and essen-         transgenic rice plants were at least 15% higher than those of the WT rice
tial regulatory process required for p21-dependent DNA damage-induced cell cycle         plants under paddy field conditions. - This work was supported by a grant
arrest.                                                                                  from the Next-Generation BioGreen 21 Program (No. PJ008115012012).

P-14                                                                                     P-17
Acceleration of Gastric Tumorigenesis Through                                            Cannabinoids Induce Pancreatic Beta-Cell Death by Directly
MKRN1-Mediated Posttranslational Regulation of p14ARF.                                   InhibitingInsulin Receptor Activation
Aram Ko1, Ji-Young Shin2, Jinho Seo1, Kang-Duck Lee2, Eun-Woo Lee1,                      Wook Kim1,*, Qizong Lao2, Yu-Kyong Shin2, Olga Carlson2, Eun Kyung
Min-Sik Lee1, Han-Woong Lee1, Il-Ju Choi2, Jin Sook Jeong4, Kyung-Hee                    Lee3, Myriam Gorospe2, Rohit Kulkarni4, and Josephine Egan2,*
Chun3, and Jaewhan Song1,*                                                               1
                                                                                          Department of Applied Chemistry and Biological Engineering, Ajou University,
 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei           Suwon 443-749, Republic of Korea, 2National Institute on Aging, NIH, Baltimore,
University, Seoul, 2National Cancer Center, Gastric Cancer Branch, Goyang-si,            MD 21224, USA, Department of Biochemistry, College of Medicine, The Catholic
Kyunggi-do, Department of Biochemistry & Molecular Biology, Yonsei University            University of Korea, Seoul 137-701, Republic of Korea, 4Department of Medicine,
College of Medicine, Seoul, Korea, 4Department of Pathology, Dong-A University           Harvard Medical School, Boston, MA 02215, USA
Medical School, Busan, Korea                                                             *Corresponding author: wookkim21@ajou.ac.kr
*Corresponding author: jso678@yonsei.ac.kr
                                                                                         Cannabinoid 1 receptors (CB1Rs) have been previously detected inpancreatic
p14ARF exerts potent tumor suppressive effects through p53 dependent or in-              β cells, where they attenuate insulinaction. We now report that CB1Rs form a
dependent pathways. We identified MKRN1 as a novel p14ARF negative E3 ligase             heteromeric complex with insulin receptor (IR) and Gαi that inhibits IR kinase
regulator with tumor suppressive effects. MKRN1 knockout mouse embryonic fibro-          activity on β cells when activated. Gαi mediates theinhibitory effects of
blasts (MEFs) exhibited premature senescence with increased p19ARF protein.              CB1Rs by direct binding to the activation loop in the tyrosine kinase domain
MKRN1 depletion in human fibroblasts or p53 defective cancer cells induced               of IR, in turn reducingphosphorylation of pro-apoptotic protein BAD and in-
p14ARF-mediated senescence and growth retardation with concurrent stabilization          ducing β-cell death. Consistently, pharmacological and geneticblockade of
of p14ARF. These effects were due to the ability of MKRN1 E3 ligase to target            CB1Rs inmouse models of diabetes leads to reduced blood glucoseand in-
p14ARF for ubiquitination and subsequent proteasome-dependent degradation.               creased β-cell survival and growth because of enhanced IR signalingthrough
Immunohistochemistry and western blot analysis of human gastric tumor tissues re-        the IRS2- AKT-BAD and -p27 pathways. This effect was not unique to pan-
vealed an inverse correlation between the two proteins. Xenograft analyses using         creatic βcells because activation of CB1Rs alsoimpeded insulin-stimulated
p53 functional AGS or dysfunctional SNU601 cells stably expressing MKRN1                 IR autophosphorylation and BAD phosphorylation in non-insulin-secreting
shRNA displayed significant growth retardation of tumors, which was reversed un-         cells. These findings provide the first evidence of direct physical and func-
der depletion of p14ARF. These findings suggest that MKRN1 could inhibit                 tional interaction between CB1R and IR signaling and provide a mechanism
p14ARF-mediated tumor suppressive processes regardless of p53 function, thus             whereby peripherally acting CB1R antagonists improve IRactivity in in-
promoting tumorigenesis.                                                                 sulin-sensitive tissues, independent of other metabolic effects of CB1Rs.

P-15                                                                                     P-18
Analysis of an Alkane-Producing Microalga, Phormidium                                    Functional Characterization of eIF4AIII as a
autumnale KNUA026 as a Potential Biofuel Feedstock                                       CBP80/20-Dependent Translation Initiation Factor
                 1,3                   2                     2                      2
Ji-Won Chang , Seung-Ik Baek , Hyun-Gu Kang , Dae-Hyun Kwon ,                            Sungjin Park, Junho Choe, Nara Oh, Kyung Mi Kim, and Yoon Ki Kim*
                  2                  2                    2               2
Won-Hyung Lee , Yong-Sun Lee , Hyung-Keun Lim , Ji-Hoon Lim ,                            School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of
                1               3                   1,3,                                 Korea, 136-701
Ho-Yeong Song , Ji-Won Hong , and Ho-Sung Yoon *
 Department of Energy Science, Kyungpook National University, Daegu 702-701,             *Corresponding author: yk-kim@korea.ac.kr
Korea, 2Gyeongsin High School, Daegu 706-819, Korea, 3ABRC, Kyungpook
National University, Daegu 702-701, Korea                                                Eukaryote translation initiation factor 4AIII (eIF4AIII), a DEAD box RNA heli-
*Corresponding author: hyoon@knu.ac.kr                                                   case and a member of the eukaryote translation initiation factor 4A (eIF4A)
                                                                                         family of translation initiation factors, is a component of the exon junction
A freshwater cyanobacterium, Phormidium autumnale KNUA026, was isolated from             complex (EJC). Only EJC is loaded onto mRNA bound by CBP80. Despite
puddles of icy water in Gyeongsan City, South Korea and its potential as biofuel         that eIF4AIII has a highly similar sequence to eIF4AI/II and previous reports
was investigated. Maximal growth was obtained when the culture was incubated at          showed eIF4AIII helicase activity in vitro, eIF4AIII is thought to have unknown
25℃ and around pH 9.0. The total lipid content of the isolate was 14.0 ± 1.4% of dry     functions in translation. Here, We suggest that eIF4AIII is involved in
weight and it was found that strain KNUA026 was able to autotrophically synthesize       CBP80/20 dependent translation (CT) for unwinding the secondary structure
heptadecane (C17H36) which can be directly used as fuel without requiring a trans-       of mRNA. On the contrary that eIF4AI/II act in eukaryote translation initiation
esterification step. This benthic cyanobacterium was also capable of forming thick       factor (eIF4E)-dependent translation (ET) for unwinding of mRNA during ribo-
mats and this self-harvesting capability may reduce the cost of harvest in commer-       some scanning, eIF4AIII is involved in CT. Furthermore, by interacting with
cial applications. Hence, P. autumnale KNUA026 appears to be promising for use in        CTIF, free cytoplasmic eIF4AIII is recruited on mRNA. This study shows that
the production of microalgae-based biofuels. - This research was funded by the           translation initiation mechanism of CT in vivo.
Global Frontier Program (2011-0031341) of the Ministry of Education, Science and
Technology (MEST), Korea.

                                                                                                                              The 24th KSMCB Winter Conference 2013        37
❱❱   Poster Presentation I

     P-19                                                                                           P-22
     Comparative Analysis of the Lipid Extraction From Energy                                       Study on Role of CIP2A in Cell Cycle Regulation
     Microalgae, Scenedesmus Obliquus
                                                                                                    Ae Lee Jeong, and Young Yang*
     Jung Yi1,3, Kyung Su Kim2, Do Hyeong Kim2, U Ju Kim2, Seong Hoon                               Center for Women's Disease, Department of Biological Science, Sookmyung
     Jeon2, Sang Heon Jeong2, Kyoung Ae Kim2, Ji Won Hong3, and Ho-Sung                             Women's University, Seoul 140-742, South Korea
     Yoon1,3,*                                                                                      *Corresponding author: yyang@sm.ac.kr
      Department of Energy Science, Kyungpook National University, Daegu, Korea,
      Gyeonggu High School, Gumi, 730-200, Korea, 3ABRC, Kyungpook National                         CIP2A is an endogenous protein phosphatase 2A interacting protein function-
     University, Daegu 702-701, Korea                                                               ing as an inhibitor of its activity. As PP2A has a critical role in not only tumor
     *Corresponding author: hyoon@knu.ac.kr                                                         suppressor activity it is conceivable that CIP2A functions as a cell cycle
                                                                                                    regulator. We investigated the expression and subcellular localization of
     Microalgae are a promising feedstock for biodiesel production. Microalgae can                  CIP2A in a cell cycle stage-specific manner. The CIP2A protein levels were
     accumulate significant amounts of lipids. The microalgal species used in the ex-               observed almost constant during cell cycle but CIP2A was found to form
     periment is Scenedesmus obliquus, one of the freshwater microalgae. These mi-                  complex with Nek2, which is a key regulator of centrosome separation and
     croalgae were used for cultivation experiments and lipid extractions in diverse                microtubule nucleation activity, through G2 to M phase. CIP2A was localized
     conditions. The studies showed that the optimal temperature and pH for S. obli-                mainly in cytoplasm, but exhibited dynamic changes in distribution, depend-
     quus are in the range of 25℃ and pH 9. S. obliquus was extracted using a mix-                  ing on the cell cycle stage. Furthermore, depletion of CIP2A using RNA inter-
     ture of chloroform and methanol (2:1). Lipid contents from S. obliquus cultured                ference prolonged mitosis time and microtubule dynamics were impaired,
     for 30 days increased to 15.8% of total biomass from 9.4% of 24 day cultures. A
     fatty acid composition analysis was performed using a gas chromatograph.                       leading to defective congression of chromosomes to the metaphase plate
     Palmitic and stearic acids were recognized as the most common fatty acids con-                 and persistent activation of the spindle checkpoint. We found that CIP2A de-
     tained in biodiesel. Therefore, these microalgae are considered to have a poten-               pletion causes mitotic delay through Nek2 stabilization and inhibiting its deg-
     tial as a substitute for fossil fuels. Nutrients such as nitrogen and phosphorus are           radation, followed by, impairing centrosome separation, microtubule
     two main components of microalgal growth and we determined the absorption                      dynamics. Thus, we propose that CIP2A is associated with cell cycle
     and lipid contents in various conditions. - This research was funded by the Global             progression.
     Frontier Program (2011-0031341) of the Ministry of Education, Science and
     Technology (MEST), Korea.

     P-20                                                                                           P-23
     Ubiquitination and Degradation of the FADD Adaptor                                             Anti-Tumoral Tctivity of TGFα-PE38 Delivered by
     Proteinregulate Death Receptor-Mediated Apoptosis and                                          Attenuated Salmonella Typhimurium
                                                                                                    Hyon E Choy*, Jae Woong Lee, Dae Jin Lim, Jae Ho Jeong, Kwang Soo
     Eun-Woo Lee1,3, Jung-Hoon Kim1, Ye-Hyeon Ahn1,4, Jinho Seo1, Aram Ko1,                         Kim, Hyong Ju Lim, Keon Hee Kim, Ju Hee Hong, Soo Mi Choy, So Ra Sin,
     Manhyung Jung1, Seok-Jun Kim2, Jae Y. Ro5, Ki-Moon Park3, Han-Woong                            and BHAT Abhay
     Lee1, Eun Jung Park4, Kyung-Hee Chun2, and Jaewhan Song1,*                                     Department of Microbiology,Chonnam National University Medical School, Hak1
      Department of Biochemistry, College of Life Science and Biotechnology, Yonsei                 dong, Dong-gu, Gwangju 501-746, Republic of Korea
     University, Seoul, Korea, Department of Biochemistry and Molecular Biology,                    *Corresponding author: hyonchoy@jnu.ac.kr
     Yonsei University College of Medicine, Seoul, Korea, 3Department of Biotechnology
     and Bioengineering, Sungkyunkwan University, Suwon, 4Cancer Immunology                         TGFα-PE38 is a recombinant fusion protein consisting of TGFα and cytosolic
     Branch, National Cancer Center, Goyang, Korea, The Methodist Hospital,                         domain of Pseudomonas exotoxin A. Results from previous clinical trials in-
     Department of Pathology and Genomic Medicine, Weill Medical College of                         dicated that this is mainly effective against the blood-bourn single cell tumors.
     Cornell University, Houston TX, USA                                                            In this study, we engineered S. typhimurium to selectively express and re-
     *Corresponding author: propadrum@gmail.com                                                     lease TGFα-PE38 out of bacteria. The engineered bacteria were targeted to
                                                                                                    implanted tumor tissue expressing high level of epidermal growth factor re-
     Fas-associated protein with death domain (FADD) is a pivotal component of death re-            ceptor (EGFR), and anti-tumor effect of TGFα-PE38 was examined. The
     ceptor-mediated extrinsic apoptosis and necroptosis. Here, for the first time, we identified   TGFα-PE38 delivered by S. typhimurium was capable of significantly sup-
     that FADD is regulated by MKRN1 E3 ligase-mediated ubiquitination and proteasomal              pressing grafted tumors. These results demonstrated a possibility of using
     degradation. MKRN1 knockdown resulted in FADD protein stabilization with rapid                 bacterial system to deliver anti-tumoral cargo proteins specifically within tu-
     death-inducing signaling complex (DISC) formation, causing hypersensitivity to extrinsic       mor tissue, resulting in selective accumulation of high level of the anti-tumor-
     apoptosis by facilitating caspase-8 and caspase-3 cleavage in response to death signals.       al proteins leading to tumor regression without significant side effect
     We also showed that MKRN1 and FADD are involved in the regulation of necrosome for-
     mation and necroptosis upon caspase inhibition. Down-regulation of MKRN1 resulted in
     severe defects of tumour growth upon TRAIL treatment in a xenograft model using
     MDA-MB-231 breast cancer cells. Suppression of tumour growth by MKRN1 depletion
     was relieved by simultaneous FADD knockdown. Our novel data reveal the mechanisms
     by which FADD is regulated via a ubiquitination-induced degradation pathway.

     P-21                                                                                           P-24
     Characterization of Histone Methylating and Demethylating                                      Astrocytic Glutamate is Critical for Synaptic Plasticity and
     Enzymes that Regulate C2C12 Myoblast Differentiation.                                          Hippocampus-Dependent Navigation
     Hansol Lee*, Young Joon Song, and Saet Byul Kim                                                Kyung-Seok Han1,4, Hyungju Park1, Jinsoo Seo2, Mijeong Park1,4, Junsung
     Department of Biological Sciences, College of Natural Science, Inha University,                Woo1,4, Suk-Hee Cho2, Stephen. F Traynelis3, Jeiwon Cho1,4, Se-Young
     Incheon, Korea                                                                                 Choi2, and C. Justin Lee1,4,*
     *Corresponding author: hlee@inha.ac.kr                                                         1
                                                                                                     Center for Neural Science and Center for Functional Connectomics, Korea
                                                                                                    Institute of Science and Technology, Seoul, Korea, 2Dept. of Physiology and
     Modifications by methyl group are known to play important roles in regulating cel-
                                                                                                    Dental Research Institute, Seoul National University School of Dentistry, Seoul,
     lular differentiation, presumably through modulating the activities of developmental
                                                                                                    Korea, 3Dept. of Pharmacology, Emory University, USA, 4Neuroscience Program,
     regulators as well as chromatin structure. In this study, we have established stable
                                                                                                    University of Science and Technology (UST), Daejeon, Korea
     cell lines in which individual H3-K9 specific KMT or KDM has been knockdowned in
                                                                                                    *Corresponding author: cjl@kist.re.kr
     C2C12 myoblast using lentiviral shRNA. We have found that knockdown of
     SETDB1 or SUV39H2 KMT resulted in complete or partial inhibition of myogenic                   Hippocampal astrocyte can release glutamatevia glutamate-permeable channels.
     differentiation, while GLP appears to promote the formation of multinucleated                  Whether this astrocytic channel-mediatedglutamate has a potency to regulate synaptic
     myotube. On the other hand, decreased expression of KDMs such as LSD1 or                       plasticity and cognitive functionremain to be determined. Here we showed a physiological
     members of JMJD2 family protein modestly inhibits myogenic differentiation except              role of astrocyticglutamate in hippocampal synaptic plasticity and learning and memory
     for JMJD2D that appears to modestly promote differentiation. NGS analysis of se-               processes,which is mediated by Ca2+-activated anion channel (CAAC), Bestrophin1
     lected cell lines have revealed that expressions of myogenic regulators as well as             (Best1) channel in the astrocytes. Astrocytic Best1-dependentglutamate in hippocampal
     genes involved in muscle differentiation are correlated with results from differ-              CA1 region could enhance a hippocampalSchaffer-collateral (SC)-CA1 synaptic response
     entiation assay. Overall, our results suggest both KMT and KDM play important                  by activating NR2A-containing synapticNMDA receptors in CA1 neurons. Moreover, the
     roles during myogenesis and we are currently investigating the direct target genes             suppression of Best1-dependentglutamate release by Best1 nullmutation produced the
     as well as possible non-histone substrates of individual KMTs.                                 impaired reversallearning and altered cue dependency in the hidden and visible platform
                                                                                                    watermaze tasks, respectively, suggesting that modulated NMDAR activity byastrocytic
                                                                                                    release of glutamate can affect cognitive flexibilities and navigationalcue dependency. Our
                                                                                                    study provides evidence that channel-mediated glutamaterelease from astrocytes is crit-
                                                                                                    ical for cognitive function.

38     Korean Society for Molecular and Cellular Biology
Poster Presentation I       ❰❰

P-25                                                                               P-28
Structural Determinants for Dynamic Regulation of Chaperone                        IL-32 Stimulates Tumorigenesis in Breast Cancer
Activity of Hsp33
                                                                                   Jeongsu Park, and Young Yang*
Yoo-Sup Lee, Tai-Geun Jung, Seo-Jin Kim, Sung-Hee Lee, Dae-Won Sim,                Research Center for Women's Disease, Department of Life Science, Sookmyung
and Hyung-Sik Won*                                                                 Women's University, Seoul, Korea
Department of Biotechnology, Research Institute for Biomedical and Health          *Corresponding author: yyang@sm.ac.kr
Science, College of Biomedical and Health Science, Konkuk University, Chungju,
Chungbuk 380-701, Korea                                                            The effects of IL-32 on the proliferation of tumors depend on its isoforms and
*Corresponding author: wonhs@kku.ac.kr                                             upon the tumor types. We here examined for the first time the role of IL-32β
                                                                                   in the MDA-MB-231 breast cancer cell line. We examined whether IL-32β af-
Hsp33, a redox-regulated molecular chaperone in prokaryotes, protects cells        fects proliferation, migration, and invasion. IL-32β expression was knocked
from an oxidative heat and/or an acute, severe oxidative stress. The oxida-        down by IL-32 siRNA and increased by using IL-32β expression vector. We
tion-induced unfolding and concomitant dimerization constitute the activation      performed the immunohistochemistry assay to examine IL-32 expression in a
process of Hsp33. In this study, solution structure of the reduced, inactive       tissue microarray containing duplicate or triplicate samples from 138 breast
Hsp33 monomer from E. coli was characterized by NMR. Although the overall          cancer patients. We found that MDA-MB-231 cells expressed only the intra-
conformation seemed to be similar to that of the previously known crystal          cellular IL-32β isoform. VEGF production was increased by the over-
structure of the reduced, dimeric Hsp33, NMR signals of specific regions           expression of IL-32β, and VEGF-mediated activation of STAT3 was respon-
were hardly detected, particularly including the N-terminal β-strand 1 and         sible for migration and invasion in MDA-MB-231 cells. Moreover, since
α-helix 1. Mutational studies implied a certain peculiar dynamics in the N-ter-    IL-32β was increased under hypoxic conditions. VEGF stimulated IL-32β
minal region and some mutations at the region resulted in the formation of         production, and the hypoxia-inducible transcription factor HIF-1α also in-
constitutively active dimer of Hsp33. Molecular dynamics simulation pre-           creased IL-32β production. These finding indicate that the IL-32β-VEGF-STAT3
dicted a beta-to-loop and a helix-to-loop conversion of the interdomain linker     or VEGF-IL-32β-STAT3 pathway plays a key role in the migration and in-
stretch, which contacts to the N-terminal region in the known crystal              vasion of breast cancer cells.
structure. Thus, it is conclusively suggested that destabilization of the linker
stretch would be the structural determinant of activation process and it is
regulated by the contacting counterpart, the N-terminal segments.

P-26                                                                               P-29
Induction of Apoptosis and Inhibition of Proliferation Activity                    Non-Structural Protein NS1 of Influenza Viruses Inhibits
in Human Breast Cancer Cells by the Ethanol Extract of Herb                        Staufen1-Mediated mRNA Decay (SMD)
                                                                                   Sang Ho Ahn, Hana Cho, Kyoung Mi Kim, and Yoon Ki Kim*
Sunyi Lee, and Young Yang*                                                         School of Life Sciences and Biotechnology, Korea University, Seoul 136-701,
Research Center for Women's Disease, Department of Life Science, Sookmyung         Republic of Korea
Women's University, Seoul, Korea                                                   *Corresponding author: yk-kim@korea.ac.kr
*Corresponding author: yyang@sm.ac.kr
                                                                                   Although the non-structural (NS) protein NS1 of influenza viruses is not es-
Herb-mixtures are a kind of traditional Korean herb. In this study, anticancer     sential for virulence, this protein is involved in host-virus interactions, viral
activity and underlying mechanisms were investigated with a herb extract us-       replication, and the translation of viral proteins. In particular, NS1 was known
ing human breast cancer MDA-MB-231 cells and MCF7. The survival rate               to interact with host protein Staufen 1 (Stau1). This interaction is important for
was reduced in a concentration- and time-dependent manner as assessed by           efficient viral replication. However, the underlying molecular mechanism, by
MTT assay. After incubation for 48h, typical apoptotic morphological changes       which NS1 has an influence on viral life cycle including replication, remains
were observed by microscope. Flow cytometry revealed that the treatment            obscure. Here, we validate using immunoprecipitation that N-terminus of
obviously induced sub-G1 arrest and apoptosis in MDA-MB-231 and MCF7               NS1, NS1(1-73) is essential for binding to Stau1. In addition, using artificial
cells. Furthermore, western blotting demonstrated down-regulation of protein       tethering system, we demonstrate that full-length NS1 and NS1(1-73) inhibit
expression of c-IAP and cleaved PARP were detected. Our results suggest            a rapid mRNA degradation triggered by tethered Stau1. Accordingly, confocal
that the extract induced cell cycle sub-G1 arrest and apoptosis in                 microscopy results reveal that, although wild-type NS1 and NS1(1-73) are
MDA-MB-231 and MCF7 cells. According to screening among 10 candidates              mainly localized to the nucleus, a significant fraction of wild-type NS1 and
of herb extracts, we found a best one in breast cancer cell lines and checked      NS1(1-73) are also detected in the cytoplasm along with Stau1. All of our re-
up anti-cancerous function itself. Therefore, we propose that one of the           sults suggest that NS1 of influenza viruses interacts with Stau1, con-
herb-extracts has potential as a breast cancer chemotherapeutic agent.             sequently inhibiting Stau1-mediated mRNA decay (SMD).

P-27                                                                               P-30
Adiponectin Deficiency in C57BL/6 Mice Suppresses EL4                              Low Magnitude Vibration Signals Inhibit the Decrease of Bone
Lymphoma Growth by Regulation of Anti-Tumor Immune Cells                           Mass in Lipopolysaccharide-Induced Mouse Model
Sora Han, and Young Yang*                                                          Tae Hyung Cho1, In Sook Kim1, Beomseok Lee1, Hoon Joo Yang1, Ji Hye
Department of Life Science, Sookmyung Women's University, Seoul, South Korea       Oh1, Su Jin Yoo1, and Soon Jung Hwang1,2,*
*Corresponding author: yyang@sm.ac.kr                                               Dental Research Institute, Seoul National University, Seoul, Republic of Korea,
                                                                                    Department of Maxillofacial Cell and Developmental Biology, Department of
We previously found that adiponectin (APN) suppresses IL-2-induced natural         Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University,
killer (NK) cell activation by down-regulating IFN-γ-inducible tumor necrosis      Brain Korea 21 2nd Program for Craniomaxillofacial Life Science, Seoul,
factor-related apoptosis-inducing ligand and Fas ligand expression. Even           Republic of Korea
though APN's anti-tumor function has been reported in several solid tumors,        *Corresponding author: ishwkim@snu.ac.kr
with few controversial results, no lymphoma studies have been conducted. In
this study, we assessed APN's role in the function of immune cells, using EL4      This study aimed to investigate the effect of vibration on the bone loss in-
and B16F10-bearing APN-knockout (APNKO) mice. We observed attenuated               duced by inflammatory cytokine, lipopolysaccharide (LPS). Balb-C mouse
EL4 growth in APNKO mice. Increased numbers of splenic NK cells were               was administered to LPS (5 mg/Kg) with two intraperitoneal injections on
found under naive conditions and splenic CTLs under EL4-challenged                 days 0 and 4. Immediately, animals were exposed to vibration (0.4 g, 45 Hz)
conditions. Splenic NK cells in APNKO mice showed enhanced cytotoxicity.           for 10 minutes per day for 4 days, and then sacrificed for micro-CT analysis
Enforced MHC class I (MHC I) expression in B16F10 led to attenuated                of bone mass. Micro-CT-based evaluation showed that LPS injection affected
growth in APNKO mice. Thus, our results suggest that EL4 regression in             the bone mass, noticeably in tibia region rather than other skeletal sites such
APNKO mice is due to not only enhanced anti-tumor immune system but also           as femur or calvarium, with significant decrease by 26 % in bone volume (BV)
to high level expression of MHC I.                                                 and by 35 % in bone mineral density (BMD). Vibratory stimulation after LPS
                                                                                   injection led to the increase of both BV and BMD by 18% and 24.5%, re-
                                                                                   spectively, compared to those of non-vibrated group, which corresponds to
                                                                                   approximately 80 % in sham control. Real time RT-PCR using bone chips ex-
                                                                                   tracted from tibia revealed the increased expression of type I collagen and
                                                                                   osteopontin genes in vibrated group more than non-vibrated group in LPS-in-
                                                                                   jected groups. These data exhibited that LPS injection induced the significant
                                                                                   loss of BV and BMD in tibia region which was suppressed by vibration.

                                                                                                                         The 24th KSMCB Winter Conference 2013          39
❱❱   Poster Presentation I

     P-31                                                                               P-34
     Structural Elucidation of the Molecular Interaction Between                        Inhibition of Adipogenesis by Protein Factors from Adult
     Tid1 and p53                                                                       Bovine Serum
     Dae-Won Sim1, Ku-Sung Jo1, Ji-Hyang Ha2, Jeong-Hwa Jang1, Seung-Wook               Jeongho Park, Hyeonwoo Tak, and Jihoe Kim*
     Chi2,*, and Hyung-Sik Won1                                                         School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of
      RIBHS, Konkuk University, Danwol-dong 322, Chungju-si, 380-701 Chungbuk-          Korea
     do, Republic of Korea, 2KRIBB, 125 Gwahak-ro, Yuseong-gu, 305-806 DaeJeon,         *Corresponding author: kimjihoe@ynu.ac.kr
     Republic of Korea
     *Corresponding author: wonhs@kku.ac.kr                                             Adipogenesis is a complex process accompanied by a variety of changes in
                                                                                        cell morphology, hormone sensitivity and gene expression. Induction of
     Tid1, an Hsp40-family protein involved in cytosolic and mitochondrial chaper-      3T3-L1 preadipocyte differentiation is possible in medium containing FBS(fe-
     on systems, as a co-chaperon of Hsp70, has been identified as a pivotal            tal bovine serum) in vitro. Differentiation of 3T3-L1 preadipocytes is com-
     component in diverse cellular processes including apoptosis. It has been re-       posed of early defferentiation mainly regulated by C/EBPβ and C/EBPδ, and
     cently suggested that Tid1 interacts with p53, leading to mitochondrial trans-     later defferentiation predominantly controlled by PPARγ(peroxisome pro-
     location of the complex and subsequent trrigering of intrinsic apoptosis. In       liferator activated recepter γ) and C/EBPα. When mitogen and hormonal
     particular, the N-terminal J-domain of Tid1 (Tid1-JD) was necessary, while ei-     stimulation initiate early differentiation, expression of C/EBPβ and C/EBPδ is
     ther N- or C-terminal domain of p53 was responsible for the interaction. Here,     increased. These regulate the expression of PPARγ and C/EBPα coopera-
     we solved the solution structure of Tid1-JD by NMR spectroscopy. The struc-        tively and adipogenesis arise as the expression of PPARγ called a master
     ture showed four conserved -heliices and a flexible HPD motif that is critical     regulator of adipogenesis. In this process, FBS is used as serum mitogen
     for the interaction to Hsp70. Then, from the chemical shift perturbations, we      that express upstream transcription factor of C/EBPβ. But differentiation of
     confirmed that the Tid1-JD directly interacts with the N-terminal TAD              3T3-L1 could not be induced when ABS is used instead of FBS. In this study,
     (transactivation domain), but not with the C- terminal domain of p53.              we showed that effect of inhibition on adipogenesis is derived from ABS
     Particularly the TAD1 of P53, rather than TAD2, provided the specific inter-       protein. We identified some protein which is exist in ABS, but not in FBS, and
     action with the Tid1-JD and phoshporylation at the TAD1 seems to promote           these proteins is potentially a inhibitor of preadipocyte differentiation.
     its binding to the Tid1-JD. These results constitute the first structural inves-
     tigation on the molecular interaction between Tid1 and P53.

     P-32                                                                               P-35
     C-terminal Truncation of a Bovine B12 Trafficking Chaperone                        Adaptor Protein Fe65 Attenuates Notch1 Signaling Via the
     Enhances the Sensitivity of the Glutathione-Regulated                              Accelerated Degradation of Notch1
                                                                                        Ji-Seon Ahn, Eun-Jung Ann, Ji-Hye Yoon, Yeong-Dae Kim, Seol-Hee Kim,
     Jinju Jeong, Jihyun Park, and Jihoe Kim*                                           Hyeong-Jin Baek, Hye-Jin Lee, and Hee-Sae Park*
     School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Korea             School of Biological Sciences and Technology, Chonnam National University,
     *Corresponding author: kimjihoe@ynu.ac.kr                                          Korea
                                                                                        *Corresponding author: proteome@jnu.ac.kr
     The human B12 trafficking chaperone hCblC is well conserved in mammals
     and non-mammalian eukaryotes. However, the C-terminal ~40 amino acids              Notch is a single-pass type Ⅰtransmembrane receptor which performs a key
     of hCblC vary significantly and are predicted to be deleted by alternative         role in the determination of cell fate, differentiation, cell proliferation, cell sur-
     splicing of the encoding gene. In this study, we examined the thermostability      vival, and cell death. Fe65 is a neuronal adaptor protein that mediates the as-
     of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and       sembly of multimolecular complexes through a variety of proteinprotein inter-
     its regulation by glutathione. t-bCblC is highly thermolabile (Tm = ~42℃) sim-     action domains: the WW domain, which binds to proline-rich sequences, and
     ilar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a     two C-terminal phosphotyrosine-binding domains. We have evaluated the
     greater extent than f-bCblC by binding of reduced glutathione (GSH) with in-       mechanism underlying the Fe65-mediated dual regulation of Notch1
     creased sensitivity to GSH. In addition, binding of oxidized glutathione           signaling. Our data show that Fe65 inhibits the transcriptional activity of
     (GSSG) destabilizes t-bCblC to a greater extent and with increased sensi-          Notch1 via an induced reduction in the protein stability of membrane-tethered
     tivity as compared to f-bCblC. These results indicate that t-bCblC is a more       Notch1, and that the level of the membrane-tethered Notch1 protein was
     sensitive form to be regulated by glutathione than the full-lengthform of the      markedly downregulated in the presence of Fe65 via the proteasomal degra-
     protein.                                                                           dation of membrane-tethered Notch1 through Itch in the cytoplasm.
                                                                                        Additionally, Fe65 interacts physically with Notch1-IC and disrupts the
                                                                                        Notch1-IC-RBP-Jk transcription complex within the nucleus.

     P-33                                                                               P-36
     Asymmetric Notch Signaling from RPE to RPC in Mouse Retina                         Wnt-5-induced CaMKII Enhances the Notch1 Signaling
                  1                                          2                     1    Pathway
     Taejeong Ha , Seong Beom Kim1, Jun Hatakeyama , Kyunghwan Moon ,
     Young-Yun Kong3,*, Kenji Shimamura2,*, and Jin Woo Kim1,*                          Eun-Jung Ann, Ji-Hye Yoon, Ji-Seon Ahn, Young-Dae Kim, Seol-Hee Kim,
      Department of Biological Sciences, Korea Advanced Institute of Science and        Hye-Jin Lee, Hyeong-Jin Baek, and Hee-Sae Park*
     Technology (KAIST), Daejeon 305-710, Korea, 2Department of Brain Morphogenesis,    School of Biological Sciences and Technology, Chonnam National University,
     Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University,        Korea
     Kumamoto 860-0811, JAPAN, 3Department of Biological Sciences, Seoul National       *Corresponding author: proteome@jnu.ac.kr
     University, Seoul 151-742, Korea
     *Corresponding author: jinwookim@kaist.ac.kr                                       Serine-threonine Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the
                                                                                        key component in noncanonical Wnt5a signaling and has been shown to reg-
     Notch signaling is critical for vertebrate neural development by maintaining       ulate its signaling. In this study, we found that CaMKII induced by Wnt5a re-
     neural progenitor cell (NPC) population and supporting glial development.          markably reduced the protein stability of the silencing mediator of retinoic
     Notch signaling is asymmetrically activated by its ligands, Delta and Jagged.      acid and thyroid hormone receptor (SMRT), a co-repressor of Notch signal-
     Notch1 is enriched at the apical part of NPC to receive ligand signal, of while    ing, through proteasomal degradation. Wnt5a was found to enhance Notch1
     sources in mammalian nervous tissues are still clearly undefined yet. To un-       intracellular domain (Notch1-IC) transcription activity, which could be in-
     derstand the sources of functionally active Notch ligands in mouse retina, we      hibited by treatment with KN93, a CaMKII inhibitor. The kinase activity of
     negatively modulated the activity of Notch ligands by specific elimination of      CaMKII was essential for the activation of Notch signaling. We also de-
     Mind bomb1 (Mib1) in retinal progenitor cells (RPCs); post-mitotic retinal neu-    termined that CaMKII could enhance the association between Notch1-IC and
     rons (PMNs); or retinal pigment epithelium(RPE). The Mib1-deficient RPCs           RBP-Jk. Furthermore, the physical association between RBP-Jk and SMRT
     are not only unable to maintain RPCs by failing to providing active ligands,       was substantially suppressed by CaMKII. We demonstrated that CaMKII di-
     but they also prematurely differentiate, suggesting that the function of Mib1 is   rectly bound and phosphorylated SMRT at Ser-1407, thereby facilitating
     essential for retinal development. We found that Mib1-enriched PMNs is not         SMRT translocation from the nucleus to the cytoplasm and proteasome-de-
     a functional source for Notch ligands, whereas the retinal pigment epithelium      pendent degradation. These results suggest that CaMKII down-regulated the
     (RPE), contacts to RPC at apical side and activates Notch in RPC. Our re-          protein stability of SMRT through proteasomal degradation.
     sults together show that asymmetric RPE-to-RPC Notch signaling plays an
     essential role in retinal development.

40    Korean Society for Molecular and Cellular Biology
Poster Presentation I       ❰❰

P-37                                                                                P-40
SGK1 Regulates the Nicastrin Degradation by its                                     Implication of Tumor Mcroenvironment on Stem-like Cancer
Phosphorylation                                                                     Cell Formation
Ji-Hye Yoon, Eun-Jung Ann, Ji-Seon Ahn, Yeong-Dae Kim, Seol-Hee Kim,                Hye Jin Kim, and Jin woong Chung*
Hyeong-Jin Baek, Hye-Jin Lee, and Hee-Sae Park*                                     Department of Biological Science, DongA University, Busan 604-714, Korea
School of Biological Science and Technology, Chonnam National University            *Corresponding author: jwchung@dau.ac.kr
*Corresponding author: proteome@jnu.ac.kr
                                                                                    Cancer stem cells (CSCs) are cancer cells that possess characteristics asso-
The gamma-secretase complex is involved in the intramembranous proteol-             ciated with normal stem cells, specifically the ability to give rise to all cell
ysis of a variety of substrates, including the amyloid precursor protein and the    types found in a particular cancer sample. CSCs may generate tumors
Notch receptor. Nicastrin (NCT) is an essential component of the gam-               through the stem cell processes of self-renewal and differentiation into multi-
ma-secretase complex and functions as a receptor for gamma-secretase                ple cell types. Such cells are proposed to persist in tumors as a distinct pop-
substrates. In this study, we determined that serum- and glucocorticoid-in-         ulation and cause relapse and metastasis by giving rise to new tumors.
duced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT.        Therefore, development of specific therapies targeted at CSCs holds hope
The SGK1 kinase activity was decisive for NCT degradation and endogenous            for improvement of survival and quality of life of cancer patients, especially
SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein             for sufferers of metastatic disease. In this study, we investigated the effect of
levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly           tumor microenvironment on CSC formation, and found that proteins from tu-
bound to and phosphorylated NCT on Ser437, thereby promoting protein                mor cells, one of the major components of tumor microenvironment, are able
degradation. Collectively, our findings indicate that SGK1 is a gamma-secre-        to induce CSC generation. Moreover, our results showed that tumor proteins
tase regulator presumably effective through phosphorylation and degradation         induced de-differentiation of normal cells, producing tumorigenic stem cells.
of NCT.                                                                             Thus, we concluded that tumor microenvironmental factors may be involved
                                                                                    in CSC formation, possibly by inducing de-differentiation of neighboring nor-
                                                                                    mal cells.

P-38                                                                                P-41
Cell Cycle-Dependent Regulation of Apoptosis                                        Inhibitory Effect of Polygala Tenuifolia on Atopic Dermatitis-like
                                                                                    Lesions Worsened by Stress
Young-Chang Cho, In-Seon Lee, and Sayeon Cho*
                                                                                                 1,2             1               1              1,2               1,2
College of Pharmacy, Chung-Ang university, Seoul 156-756, Korea                     Bongjun Sur , Bombi Lee , Mijung Yeom , Insop Shim , Hyejung Lee ,
*Corresponding author: sycho@cau.ac.kr                                                                    2,
                                                                                    and Dae-Hyun Hahm *
                                                                                     Acupuncture and Meridian Science Research Center, Kyung Hee University,
Mitogen-activated protein kinase (MAPK) signaling pathway plays a key role          Hoegi-dong, Dongdaemoon-gu, Seoul, 130-701, Korea, 2Department of Basic
in cell growth, differentiation, and apoptosis. One of the crucial biological re-   Oriental Medical Science, College of Oriental Medicine, Kyung Hee University,
sponses mediated by stress-activated MAPK pathways appears to decide                Hoegi-dong, Dongdaemoon-gu, Seoul, 130-701, Korea
cell fate by regulating apoptosis. Apoptosis signal-regulating kinase 1 (ASK1)      *Corresponding author: dhhahm@khu.ac.kr
is a member of MAP kinase kinase kinase family and plays a critical role in
cellular stress-induced apoptosis. ASK1 is activated by phosphorylation at          Many skin disorders, such as atopic dermatitis (AD) can be exacerbated by
Thr-838 and is suppressed by phosphorylation at Ser-83 or Ser-967. Here,            various psychological stresses. Increasing evidence suggests that it is asso-
we show that cell division cycle 25 homolog C (cdc25C) dephosphorylates             ciated with the degranulation of mast cell via CRF released by stress. The
phospho-Thr-838 and inhibits ASK1 activity. In cells arrested in G2/M phase         aim of this study is to assess the effects of Polygala tenuifolia Willdenow
with nocodazole, phosphorylation at Thr-838 of ASK1 was elevated, while             (PTW) on stress-induced exacerbation of AD in trimellitic anhydride
phosphorylation at Ser-83 and Ser-967 was decreased. In vitro phosphatase           (TMA)-induced AD mice model. AD was induced by repeated application of
assays showed that cdc25C directly dephosphorylates phospho-Thr-838. In             TMA in BALB/c mice. For stress-induced exacerbation of AD, animals were
addition, small interfering RNA (siRNA) analysis showed that knock-down of          repeatedly exposed to immobilization (6 days). PTW was administered at the
cdc25C increased ASK1 kinase activity. Furthermore, ASK1-mediated and               dose of 50 or 250 mg/kg once daily for 6 days. The efficacy of PTW was eval-
caspase-3/9-dependent apoptosis was reduced by cdc25C expression. Thus,             uated by assessing scratching behavior, serum IgE level, and skin in-
our results suggest that cdc25C negatively regulates ASK1 during G2/M               flammation such as ear thickness and the expression of proinflammatory
phase transition                                                                    mediators. Immobilization stress worsened the symptoms of AD such as
                                                                                    scratching behavior and skin inflammation. PTW significantly reduced
                                                                                    scratching behavior, serum IgE, and the skin inflammation, which increase
                                                                                    with stress-induced exacerbation of AD. These results suggest the effective-
                                                                                    ness of PTW for prevention or the treatment of the diseases that is linked to
                                                                                    stress-related immune dysfunction.

P-39                                                                                P-42
Anti-tumor Effect of Proteins from Gecko on Human Cervical                          Effect of Fucoidan Against Poloxamer 407-induced
Cancer Cells via PI3 kinase-Akt Pathway                                             Hyperlipidemia Through the Modulation of SREBP-2 in
                                                                                    C57BL/6 Mice
Aejin Jeong, and Jinwoong Chung*
Department of Biological Science, Dong-A University, Busan 604-714, Korea           Jinhee Park1, Mijung Yeom1, Bombi Lee1, Insop Shim1,2, Hyejung Lee1,2,
*Corresponding author: jwchung@dau.ac.kr                                            and Dae-Hyun Hahm1,2,*
                                                                                     Acupuncture and Meridian Science Research Center, Kyung Hee University,
Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells,                                                               2
                                                                                    Hoegi-dong, Dongdaemoon-gu, Seoul, 130-701, Korea, Department of Basic
and its signaling mechanisms were assessed by viable cell counting, propi-          Oriental Medical Science, College of Oriental Medicine, Kyung Hee University,
dium iodide (PI) staining, and Western blot analysis. GP induced the cell           Hoegi-dong, Dongdaemoon-gu, Seoul, 130-701, Korea
death of HeLa cells in a dose-dependent manner while it did not affect the vi-      *Corresponding author: dhhahm@khu.ac.kr
ability of normal cells. Western blot analysis showed that GP decreased the
activation of Akt, and co-administration of GP and Akt inhibitors synergisti-       Hyperlipidemia is considered one of the major risk factors in causing car-
cally exerted anti-tumor activities on HeLa cells, suggesting the involvement       diovascular diseases such as arteriosclerosis. In the present study, the in-
of PI-3 kinase/Akt pathway in GP-induced cell death of the cancer cells.            hibitory effects of fucoidan on hyperlipidemia was investigated in the polox-
Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by over-        amer 407 (P407)-induced mouse model of hyperlipidemia. To develop an ani-
expression of constituvely active form of Akt in HeLa cells. The candidates of      mal model of acute hyperlipidemia, P407 at a dose of 250 mg/kg was intra-
the functional proteins in GP were analyzed by Mass-spectrum. Taken to-             peritoneally injected into the mice. Fucoidan at doses of 10, 30, or 50 mg/kg
gether, our results suggest that GP elicits anti-tumor activity against HeLa        were treated 2 h after P407 injection. Serum levels of total cholesterol (TC),
cells by inhibition of PI-3 kinase/Akt pathway.                                     triglyceride (TG) and high-density lipoprotein-cholesterol (HDL-c) were ana-
                                                                                    lyzed 24 h after P407 injection. Atherogenic index (AI) was calculated. The
                                                                                    mRNAs and proteins of SREBP-2 and HMGCR in the liver were determined
                                                                                    using RT-PCR and West blot analysis, respectively. Fucoidan treatment
                                                                                    dose-dependently inhibited P407-induced increases in the serum levels of
                                                                                    TC, TG and AI, whereas remarkably increased HDL-c level. Fucoidan also
                                                                                    dose-dependently inhibited the mRNA and protein expression of SREBP-2
                                                                                    and HMGCR in the liver. Our study suggests that fucoidan could be used as
                                                                                    a useful agent to prevent or to treat Hyperlipidemia.

                                                                                                                          The 24th KSMCB Winter Conference 2013         41
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