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05F034
O-13
The adenosine A3 receptor agonist, Cl-IB-MECA, induces cell death through
ROS-dependent down-regulation of ERK and Akt in human glioma cells
Thae Hyun Kim, Ji Yeon Park, Chae Hwa Kwon, Yong Keun Kim and Jae Suk
Woo
Department of Physiology, Pusan National University School of Medicine,
Yangsan, 626-770, Korea
Adenosine A3 receptor (A3AR) is coupled to G proteins that are involved in a
variety of intracellular signaling pathways and physiological functions. The
A3AR agonist, 2-chloro-N6-(3-iodobenzyl) adenosine-5’-Nmethylcarboxamide (Cl-IB-MECA), has been reported to induce cell death in
various cancer cells. However, the effect of CI-IB-MECA on cell viability in
glioblastoma is still poorly understood. Therefore, this study was undertaken to
examine the effect of CI-IB-MECA on glioma cell viability and to determine its
underlying mechanism. CI-IB-MECA caused reduction in cell viability in a
dose- and time-dependent manner. Treatment of CI-IB-MECA induced an
increase in intracellular Ca<sup>2+</sup> followed by enhanced reactive
oxygen species (ROS) generation. EGTA and N-Acetylcystein (NAC) blocked
the cell death induced by CI-IB-MECA, suggesting that Ca<sup>2+</sup> and
ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis
showed that CI-IB-MECA induced a decrease in extracellular signal-regulated
kinases (ERK1/2) and Akt. Transfection of constitutively active forms of MEK,
the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA
increased caspase-3 activation and the CI-IB-MECA-induced cell death was
blocked by caspase inhibitor. Collectively, these findings demonstrate that CIIB-MECA induce caspase-dependent cell death through down-regulation of
ERK and Akt in glioma cells. The data of the present study suggest that A3AR
agonists may be used as a preventive and therapeutic agent for glioma
malignant.
Key Word : A3AR, Cl-IB-MECA, human glioma cell
저자 : 김태현, 박지연, 권채화, 김용근, 우재석
소속 : Department of Physiology, Pusan National University School of Medicine,
Yangsan, 626-770, Korea
05F042
O-14
Hypoxia-inducible factors are positively regulated by NEDDylation of alpha
subunits
Ji-Hye Ryu¹³, Hyoung-Sook Park¹, Jong-Wan Park²³, Yang-Sook Chun¹³
Departments of ¹Physiology and ²Pharmacology, ³Ischemic/Hypoxic Disease
Institute, Seoul National University College of Medicine, 28 Yongon-dong,
Chongno-gu, Seoul 110-799, Korea
Hypoxia-inducible factor alpha proteins (HIF-αs) are regulated oxygendependently, and transactivate numerous genes essential for cellular
adaptation to hypoxia. NEDD8, a member of the ubiquitin-like family, covalently
binds with its substrate proteins, and thus, regulates their stabilities and
functions. In the present study, we examined the possibility that the HIF
signaling is regulated by NEDD8. It was found that HIF-1α expression and
activity were inhibited by knocking-down APPBP1 E1 enzyme for neddylation,
and stimulated by NEDD8. In addition, HIF-1α and α were stabilized and
covalently modified by NEDD8, which also inhibited the ubiquitination and
proteasomal degradation of HIF-1α. Because, HIF-1α neddylation and
stabilization were observed in VHL(-/-) and in p53(-/-) cells, it appears that
NEDD8 stabilizes HIF-1α by inhibiting a ubiquitin-proteasome system other
than the PHD/VHL-dependent oxygen-sensing system. Based on these
findings, we propose a novel role for neddylation in HIF signaling. Furthermore,
given the positive role played by HIF-1αs in tumor growth, we suggest that the
targeting of neddylation be viewed as a potential means of treating malignant
tumors expressing high levels of HIF-αs.
Key Word : Hypoxia-inducible factors, NEDD8, post-translational modification,
proteolysis
저자 : 류지혜¹³, 박형숙¹, 박종완²³, 전양숙¹³
소속 : 서울대학교 의과대학 생리학교실
05F056
O-15
Cholesterol enrichment manifests cellular changes associated with Alzheimer’s
disease–linked presenilin mutations
Yoon Sun Chun, Sungkwon Chung
Department of Physiology, Sungkyunkwan University School of Medicine, Suwon
440-746, Korea
Our previous study shows that mutations in the presenilin genes (PS1, PS2),
the leading cause of autosomal dominant familial Alzheimer’s disease (FAD),
cause down-regulation of phosphatidylinositol 4,5-bisphosphate (PIP2). This
affects the generation of toxic β-amyloid peptides (Aβ) as well as the activity
of Mg2+-inhibited cation (MIC) channel. We also have shown that increased
cholesterol level down-regulates PIP2, inhibiting not only PIP2-sensitive
endogenous MIC channel but also exogenously expressed PIP2-sensitive K+
channels. Thus, we tested whether cholesterol enrichment induces the same
cellular changes associated with FAD-linked PS mutations. Elevated cholesterol
level increased the production of longer more fibrillogenic forms of Aβ, and
induced the deregulation of Ca2+ signaling including the increase of agonistinduced ER Ca<sup>2+</sup> response as well as the decrease of
capacitative Ca<sup>2+</sup>2+ entry (CCE). Intracellular delivery of PIP2
reversed these cholesterol induced changes. We found that cholesterol
increased the expression of some phospholipase C (PLC) isoforms, such as
PLCβ1. These results suggest that cholesterol enrichment, and FAD-linked PS
mutations share the same molecular mechanism, i.e. the down-regulation of
PIP2, which may serve as the molecule linking cholesterol metabolism to the
pathogenesis of FAD.
Key Word : Alzheimer’s disease, cholesterol, PIP2
저자 : 천윤선,정성권
소속 : 성균관대학교 의과대학 생리학교실
05F063
O-16
Chondrogenic differentiation of human clonal mesenchymal stem cells using
CoCl₂
Mi Hyun Lim¹, Byung Hyune Choi², A Rum Seo¹, Byoung-Hyun Min³, So Ra
Park¹*
Department of Physiology¹, Division of Biomedical and Bioengineering
Sciences², Inha University College of Medicine, Incheon, Department of
Orthopedic Surgery³, School of Medicine, Ajou University, Suwon, Korea
Human mesenchymal stem cells (hMSCs) obtained from bone marrow have a
potential to differentiate into multiple cell lineages including the chondrocyte.
Although various methods for differentiating MSCs into the chondrogenic
lineage have been reported, it is still necessary to improve the efficiency of
differentiation. We investigated the effects of hypoxia mimicking chemicals
(CoCl₂ and desferroxamine) on chondrogenic differentiation of human clonal
MSC established by the preplate technique The hcMSC at passage 2 were
encapsulated in alginate layer and cultured 3-dimensionally (3-D) in
chondrogenic defined media in the presence of 10 ng/ml TGF-β or 5μM
CoCl₂(or desferroxamine) for 2 weeks. The chondrogenic differentiation of
hcMSC was evaluated by RT-PCR analysis for chondrogenic marker genes
such as type II collagen, Sox9, and aggrecan. The safranin-O staining for
sulfated proteoglycans was also performed. CoCl₂ (or desferroxamine) clearly
induced chondrogenic differentiation of hcMSC as efficiently as TGF-β, a wellknown chondorgenic factor. However, treatment of CoCl₂ and TGF-β at the
same time showed no additive effect. When the levels of hypoxia-inducible
proteins of HIF-1α and VEGF were examined, CoCl₂ induced the levels of
both proteins but TGF-β induced that of HIF-1α alone. In conclusion CoCl₂, a
hypoxia mimicking chemical could be an efficient and cheaper alternative to
TGF-β for the chondrogenic differentiation of hMSCs in vitro.
Key Word : Human mesenchymal stem cells, CoCl₂, TGF-β
저자 : 임미현¹, 최병현², 서아름¹, 민병현³, 박소라¹*
소속 : Department of Physiology Inha University College of Medicine, Incheon,
400-712, Korea
05F155
O-17
N-acetylcysteine inhibits RhoA and promotes apoptotic cell cearance during
intense lung inflammation
Changsuk Moon, Ye-Ji Lee, Hyun-Jeong Park, Jihee Lee
Department of Physiology, Division of Cell Biology, Ewha Medical Research
Center, School of Medicine, Ewha Womans University, Seoul, South Korea
Rationale: The resolution of pulmonary inflammation seen in various
inflammatory lung conditions depends on the clearance of apoptotic cells to
prevent permanent tissue damage or progressive disease. Uptake of apoptotic
cells by alveolar macrophages is suppressed by oxidants through the activation
of Rho signaling. Objective: We hypothesized that antioxidant exposure would
increase the ability of alveolar macrophages to clear pulmonary apoptotic cells
through the inhibition of RhoA. Methods: The effects of the antioxidant Nacetylcysteine (NAC) on the pulmonary immune response were seen in mice
treated intratracheally with LPS, LPS+NAC, or saline. Apoptotic cell clearance,
RhoA activity, and changes in the lung inflammatory responses were analyzed
in vivo or ex vivo. Results: Neutrophil accumulation, apoptosis, and oxidant
production peaked at 3 d post-LPS treatment. NAC enhanced the clearance of
apoptotic cells and inhibited RhoA activity in alveolar macrophages at 3 d
post-LPS treatment. NAC suppressed LPS–induced proinflammatory mediators,
enhanced the production of TGF-β1, reduced the accumulation of
inflammatory cells, and reduced protein levels in BAL fluid. In the presence of
ex vivo apoptotic cells, alveolar macrophages exposed to LPS or LPS+NAC had
reduced TNF-α levels and increased TGF-β1 levels. A Rho kinase inhibitor
mimicked the effects of NAC on the clearance of apoptotic cells, the
production of proinflammatory mediators, and TGF-β1 upregulation.
Conclusions: These results indicate that NAC can expedite the resolution of
LPS-induced pulmonary inflammation through the inhibition of RhoA activity
and the enhancement of apoptotic cell clearance.
Key Word : NAC, efferocytosis, inflammation, RhoA
저자 : 문창숙, 이예지, 박현정, 이지희
소속 : 이화여자대학교 의과대학 생리학 교실
05F129
O-18
Microarray analysis of gene expression in quinolone antibiotics-treated
cardiomyocytes
Kyung-Jong Won1, Chang-Kwon Lee1, Haiyue Lin1, Jun Ho Kim1, Seung Hyo
Jung1, Soo Hyun Jung1, Soo Min Cho2, Young Min Bae1, Ho-Chul Shin2, Hwan
Myung Lee3, Bokyung Kim1
1Department of Physiology, Konkuk University School of Medicine, Chungju 380701; 2Department of Veterinary Pharmacology and Toxicology, College of
Veterinary Medicine, Konkuk University, Kwangjin-ku, Seoul 143-701, Korea;
3Department of Herbal Cosmetic Science, College of Natural Science, Hoseo
University, Asan 336-795, Korea
This study investigated the gene expression profile in cardiomyocytes treated
with quinolone antibiotic agents, ciprofloaxin, ofloxacin, and sparofloxacin,
which have been reported to induce side effects on cardiac function, using the
GeneChip analysis performed with the Whole Rat Genome Oligo Microarray.
Antibiotics-treated cardiomyocytes showed that the expressions of 4,263 gene
probes were upregulated and the expressions of 5,556 genes were downregulated in comparison with DMSO-treated control cardiomyocytes. Of them,
the number of 7-fold increased expressions in the treatments of sparofloxacin,
ofloxacin, ciprofloxacin and enoxacin was 233, 114, 77 and 88 genes,
respectively, and that of 20-fold increased ones was 11, 4, 3 and 5 gene
probes compared with control, respectively. Moreover, the treatment of
sparofloxacin, ciprofloxacin and ofloxacin to cardiomyocytes showed 7-fold
increased expressions of 19 gene probes compared with that of enoxacin that
has not been known to have a side effect on cardiac functions. These 19 gene
expressions increased by the treatment of 3 antibiotics were found to have a
similar tendency in their differences compared with enoxacin-induced gene
expression changes. These data may provide the candidates of indicator
predicting antibotics-induced cardiovascular disorders. This research was
supported by a grant (08172KFDA508) from Korea Food & Drug Administration
in 2008.
Key Word : Quinolone, antibiotics, neonatal cardiomyocytes, DNA chip
저자 : 원경종 1, 이창권 1, Haiyue Lin1, 김준호 1, 정승효 1, 정수현 1, 조수민 2,
배영민 1, 신호철 2, 이환명 3, 김보경 1
소속 : 건국대학교 의학전문대학원 생리학교실