Medical Technology (13:30 ～16:00 THP Seminar Room)
Wakusawa
Session chairs: Prof. Shinya
Sinya Wakusawa
Prof. Tsutomu Kawabe

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Sleep disorder and sleep-disordered breathing in the elderly
C.Nakazaki1), A.Noda2), N.Fujita3), S.Yamada1), 4), Y.Koike2), S.Wakusawa1), 4)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of
Medicine, Nagoya, Japan
2) College of Life and Health Sciences, Chubu University3) Department of Clinical Laboratory at Nagoya University Hospital4) Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan

Introduction
The elderly spend more time in bed, experience more
awakenings during the night,1) and have increased complaintsof sleep disorder such as the difficulty initiating sleep,
difficulty remaining asleep and early morning awakenings.2)
Elderly individuals complain primarily about sleep disorder,
which are often secondary to other disorders. 1)
Aging substantially increases the risk of obstructive sleep
apnea syndrome (OSAS),3) and OSAS is associated withhypertension, cerebrovascular disorder, cardiovasculardisease and all-cause mortality.4) However, effects of aging
on pathophysiology of apnea/hypopnea in clinical settings
have not been clearly investigatedWe assessed the sleep disorder in middle-aged andelderly people, and examined the relation between sleep
disordered-breathing (SDB) and cardiovascular function in
the elderly.
Materials and Methods
Subjects: 32 middle-aged subjects (51.3±4.1 years, male:
16 subjects, female: 16 subjects) and 20 elderly (71.7±5.8
years, male: 5 subjects, female: 15 subjects) were targeted.
The study protocol was approved by the appropriate
institutional review committee at the Nagoya University, andinformed written consent from the subjects was obtained at
the beginning of the assessment.
Functional Outcome Measures: Subjective sleep
symptoms, disturbances, and patterns were assessed with thePittsburgh Sleep Quality Index (PSQI). The total global score
ranges from 0 to 21, and greater scores indicate higher levelsof sleep symptoms.5) Subjects with PSQI ≥ 6 were defined as
insomnia. Daytime sleepiness was quantified using the
Epworth Sleepiness Scale (ESS). Possible scores ranges from0 to 24, and higher scores reflect greater sleepiness. A cut-off
value ≥ 11 indicated excessive daytime somnolence (EDS) 6).
Actigraphy: The actigraph (Ambulatory Monitoring, Inc,
USA) was worn around the wrist of a non-dominant hand.
The actigraph data were assessed using the algorithm
supplied by the ActionW-2 clinical sleep analysis software
package for Windows (Ambulatory Monitoring, Inc, USA).
Sleep and activity were scored according to the Cole-Kripke
formula.7) Total sleep time (TST) was measured using
actigraphy.8)
Measurements for Sleep Apnea: To assess the sleep
apnea, overnight examination was performed on eachsubjects using the portable home monitoring (apnomonitor:
CHEST) and we calculated apnea-hypopnea index/h (AHI/h),
oxygen desaturation index/h (ODI/h) and lowest arterial
oxyhemoglobin saturation (Lowest SpO2). We determined
the subjects of AHI ≥ 5/h as sleep apnea syndrome (SAS).

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Markers of Atherosclerosis: We evaluated atherosclerosis
from Pulse Wave Velocity (PWV) and carotid ultrasonography.
PWV was measured using a volume-plethysmographic (from
PWV/ABI; Nihon Colin Co.,Ltd.,, Komaki, Japan), which
simultaneously records brachial-ankle PWV (baPWV), bloodpressure, an electrocardiogram, and heart sounds.9) The intimamedia thickness (IMT) of the carotid arteries was estimated by
B-mode ultrasound (HEWLETT PACKARD SONOS 2000：
PHILIPS) with a 7.5MHz. 10) 11), 12)
Echocardiograph: Standard echocardiography including
measurement of transmitral flow velocity indices (HEWLETT
PACKARD SONOS 2000 ： PHILIPS) was performed in all
subjects. Left atrium dimension (LAD), left ventricular enddiastolic dimension (LVDd) and left ventricular end-systolicdimension (LVDs) were measured by two-dimensional M-mode,and the left ventricular ejection fraction (LVEF) was calculated.
The peak early velocity (E) and atrium contraction velocity (A),their ratio (E/A) and the deceleration time of peak E velocity(DcT) were measured from mitral inflow velocities.
Data analysis: All statistical analyses were performed using
a statistical software package (Statview for Windows version
4.54 SAS Institute Inc; Cary, NC). Data were presented bymean ± standard deviation (SD), and analysis of variance follow(ANOVA) by Scheffe’s test was used to compare means ofcontinuous values between subgroups. A probability (p) value <0.05 were considered statistically significant.
Results
ESS was significantly higher in the middle-aged groupthan in the elderly group (8.1±5.1 vs 3.9±2.9, p=0.01). (Figure1)
Bed time was significantly earlier in the elderly group thanin the middle-aged group. (21:25±0.19 vs 22:59±0.17, p=0.019)
There was no significantly difference between two groups inwake-up time. (Figure 2)
The prevalence of PSQI ≥ 6 (insomnia) was 45% in theelderly group, and it was 28% in the middle-aged group. In theelderly, Time in bed was significantly extended in the insomnia
group than in the non-insomnia group. (8.6±1.6h vs 7.5±1.0h,
p=0.038) (Table 1)
There were no significantly differences between SAS andnon-SAS groups in atherosclerotic and echocardiographic
parameters. (Table 2)

Discussion
Epidemiological studies have implicated OSAS as a risk
factor for the development of cardiovascular andcerebrovascular disease. However, in this study, cardiovascularfunction did not significantly differ between SAS and non-SASgroups in the elderly. Moreover, AHI did not significant changewith a presence of hypertension or hyperlipidemia.
ESS score was significantly higher in the middle-aged
group than in the elderly group, and bed time was significantly
later. Daytime sleepiness was found in the middle-aged groupand TST during the night was short. In the elderly, daytime
sleepiness was not detected, but insomnia was permitted
especially who spend long time in bed. Sleep disorder may
relate to decrease of daytime activity and sleep quality.
In conclusion, comparison of sleep disorder/SDB andcardiovascular function between middle-aged and elderly
population may provide insight into the pathophysiology ofsleep disorder/SDB associated with aging.

p=0.010

14
12

ESS

10
8
6
4
2
0
middle-agedelderly

Figure 1. ESS between middle-aged and elderly group
ESS: Epworth Sleepiness Scale

middle-aged

p=0.019elderly
0：00

6：00

12：00 18：00 24：00

6：00

Bed time

middle-agednselderly
0：00

6：00

3：00

9：00

Wake-up time

Figure 2. Bed time and wake-up time between middle- aged
and elderly group
Table 1. Time in bed in the elderly group

insomnia

non-insomnia

n=9

n=11

7.2±2.4

4.3±1.2<0.001Time in bed (hr) 8.6±1.6

7.5±1.0

<0.05

PSQI

P value

Data are presented as mean ± SD.
PSQI: Pittsburgh Sleep Quality Index
Table2 . Atherosclerotic and echocardiographic
parameters in the elderly group

R-IMT Max (mm)
L-IMT Max (mm)R-baPWV (cm/s)
L-baPWV (cm/s)
LVEF (%)
E/A
DcT (sec)SAS

non-SAS

n=11

n=9

P value

1.1 ± 0.5
1.0 ± 0.3
1568.0 ± 285.0
1597.0 ± 256.0
70.0 ± 12.1
0.9 ± 0.3
210.0 ± 59.4

ns
ns
ns
ns
ns
ns
ns

0.9 ± 0.4
0.9 ± 0.5
1608.0 ± 382.0
1628.0 ± 300.0
66.4 ± 6.1
0.8 ± 0.2
207.0 ± 36.5

Data are presented as mean ± SD.
IMT: intima-media thickness; baPWV: brachial-ankle pulse
wave velocity; LVEF: left ventricular ejection fraction; E/A:
peak early systolic velocity (E)/atrial contraction velocity(A); DcT: deceleration time of peak E velocity

References[1] Sonia Ancoli-Israel, J. Steven Poceta, et al. Identificationand treatment of sleep problems in the elderly. Sleep Med
Rev 1997; 1: 3-17
[2] Ohayon MM, et al. Epidemiology of insomnia: what weknow and what we still need to learn. Sleep Med Rev 2002;
6(2):97–111.
[3] Matthias E, Amy S. Jordan, Nancy L. Chamberlin, et al. The
influence of aging on pharyngeal collapsibility during sleep.
Chest 2007; 131: 1702-1709[4] Heather E, David J, et al. Neuropsychological function in
obstructive sleep apnoea. Sleep Med Rev 1999; 1: 59-78[5] Buysse DJ, Reynolds CF, 3rd, Monk TH, et al. The
Pittsburgh Sleep Quality Index: a new instrument forpsychiatric practice and research. Psychiatry Res 1989; 28:
193-213[6] Johns MW, et al. A new method for measuring daytime
sleepiness: the Epworth sleepiness scale. Sleep 1991; 14(6);540-545[7] Cole RJ, Kripke DF, Gruen W, Mullaney DJ, Gillin JC:
Automatic sleep/wake identification from wrist activity.
Sleep 1992; 15: 461-469[8] Van Den Berg JF, Van Rooij FJ, Vos H, et al. Disagreement
between subjective and actigraphic measures of sleep
duration in a population-based study of elderly persons. J
Sleep Res. Sep 2008;17(3):295-302[9] Yufu K, Takahashi N, Hara M et al. Measurement of thebrachial-ankle pulse wave velocity and flow-mediateddilatation in young, healthy smokers. Hypertens Res 2007;30(7); 607-612[10] Kitamura A, Iso H, Imano H, et al. Prevalence andcorrelates of carotid atherosclerosis among elderly Japanesemen. Atherosclerosis 2004; 172:353-359[11] Renzhe Cui, Kitamura A, Yamagishi K, et al. Ankle-arm
blood pressure index as a correlate of preclinical carotid
atherosclerosis in elderly Japanese men. Atherosclerosis
2006; 184: 420-424[12] Handa N, Matsumoto M, Maeda H, et al. Ultrasonicevaluation of early carotid atherosclerosis. Stroke 1990; 21;1567-1572
Author address
E-Mail: nakazaki.chie@h.mbox.nagoya-u.ac.jp

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Mycobacterium tuberculosis-induced expression of Granulocyte-Macrophage
Colony Stimulating Factor is mediated by ERK1/2 MAPK or PI3-K/Akt signaling
pathwayJang-Eun Cho1), Sang-Jung Park1), Yong-Mi Kim2), Sang-Nae Cho2) , Hye-Young Lee1) and Yoon-Suk Kim1)
1) Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju
2) Department of Microbiology, College of Medicine, Yonsei University, Seoul

Introduction
Mycobacterium tuberculosis, the causative agent oftuberculosis, has already infected nearly a third of the world’s
population causing approximately 2 million deaths yearly.
Infection with MTB affects the recruitment and activation of
circulating effector leukocytes by influencing induction andsecretion of cytokines from infected macrophages. Infected
macrophages release a variety of inflammatory cytokines as
defense mechanisms against MTB. The cytokine GM-CSF
plays an important role in the differentiation of monocytes,
alveolar macrophages and dendritic cells. GM-CSF acts locallyas a proinflammatory cytokine. In the lungs, GM-CSF is very
important for macrophage maturation and differentiation andhost defense. It was previously reported that GM-CSF can
induce up-regulation of MHC class II and costimulatory
molecules such as CD80 and CD86 on antigen presenting cells(APCs) as well as increasing their phagocytic activity andstimulatory capacity. When this cytokine is lacking, as in genedisrupted mice, the architecture of the lung is altered andalveolar macrophages become foamy in appearance. In addition,these cells have defects in phagocytosis, bacterial killing, as
well as loss of Toll-like receptor expression and signaling. In
the context of tuberculosis, GM-CSF may also contribute to thecytokine/chemokine milieu responsible for granuloma
formation in the lung. Over-expression of GM-CSF in the lungs
impairs protective immunity against MTB and therefore careful
regulation of pulmonary GM-CSF levels may be critical insustaining protection against chronic tuberculosis disease. GMCSF act as a pivotal factor for the development of alveolar
macrophages in lung. Increasing GM-CSF levels into thechronic stage of the infection results in elevated cellproliferation and phagocytic activity. GM-CSF regulates both
pulmonary surfactant homeostasis and the differentiation andproliferation of functionally competent alveolar macrophages.
But the intracellular signaling pathways involved in theregulation of GM-CSF expression in alveolar macrophages areunclear. In this study, we aimed to elucidate the involvement of
GM-CSF in the host immune response against MTB infection.
We observed mycobacterium-stimulated induction of GM-CSF
and evaluated the associated with signal transduction pathway.
Materials and Methods
Preparation of Mycobacteria
Mycobacterium tuberculosis H37Rv (ATCC 27294), M.
bovis BCG (ATCC 35748) and clinical isolated Beijing family
of MTB strain (K-strain) obtained at Yonsei Medical school in
Seoul and used in this study was grown for about four weeks at
37°C as a surface pellicle on Sauton medium enriched with
0.4% sodium glutamate and 3.0% glycerol. The surface
pellicles were collected and disrupted by gentle vortexing with
6mm glass beads. After clumps had settled, the upper
suspension was collected and aliquots were stored at -80°C.
Before infection,

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aliquots were thawed and quantitated for viable colony-forming
units (CFU) on Middlebrook 7H10 agar (Difco, USA)..
Cell culture and infection of mycobacteria
THP-1 cell line was maintained in RPMI 1640 medium
with 2 mM glutamine, 10% heat inactivated fetal bovine serum,
100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL,USA) at 37℃ under 5% CO2 condition. THP-1 cells wereseeded in six-well plates and treated with 50 nM phorbol-12myristate-13-acetate (PMA; Sigma) for 48 h to induce
differentiation into macrophage-like cells, then washed threetimes with antibiotics free RPMI 1640 medium. Before
infection, differentiated THP-1 cells were reconstituted in
antibiotics free RPMI 1640 medium with 5% FBS. Cells wereincubated with MTB at 10 MOI for 0, 1, 1.5, 3, 6, 9, 12, 18, 24,
48 or 72 h. PMA-differentiated THP-1 cells were pretreatedwith inhibitors for 45 min before stimulation with MTB H37Rv
for 4 h at 10 MOI.
RNA extraction and Semi-quantitative RT-PCR
Total RNA was extracted from cultured cells using Trizol
Reagent (Invitrogen, USA) according to the manufacturer’s
instruction. cDNA was synthesized by reverse transcription
with 2 μg total RNA, 0.25 μg of random hexamer (Invitrogen)and 200 unit of Murine Molony Leukemia Virus Reverse
Transcriptase (MMLV-RT; Invitrogen) for 10min at 25°C,
50min at 37°C and 15min at 70°C. Diluted reverse-transcribed
cDNA was used subsequent 25 μl PCR amplification. PCR
amplification using 0.2 units of G-Taq polymerase (Cosmo
Genetech, Korea) was performed in a thermocycler (Applied
Biosystems, USA) for 35 cycles using GM-CSF primer.
GAPDH was ampilifed as an internal control. The intensity ofeach band amplified by RT-PCR was analyzed using Gel Doc
EQ Quantity One (Bio-rad, Italy) and normalized to GAPDHmRNA in corresponding samples.
Enzyme-linked immunosorbent assay (ELISA)
Cell culture supernatants from MTB-infected THP-1 cells
were collected 24 h after infection. Cell culture supernatants
were analyzed using Human GM-CSF ELISA MAX™
Standard (BioLegend, USA), as recommended by the
manufacturer.
Statistics
All values are given as mean ± standard deviation (SD).
When a significant difference was detected, further analysis
was performed using a Student’s t-test. A p value of less than
0.05 was considered significant.
Results
MTB infected THP-1 cells induced a dose-dependent mannerand a time-dependent increase in GM-CSF mRNAExpression of GM-CSF was up-regulated by infection of
MTB in a dose-dependent manner, reaching a peak at 20 MOI
of MTB. We also examined the effects of mycobacterial
infection on the timing of GM-CSF expression. GM-CSF
expression was up-regulated in response to MTB in a timedependent manner. The mRNA level of GM-CSF peaked at 6 hafter mycobacterial infection, and then declined gradually for
72 h.
Induction of GM-CSF by MTB is mediated by p38 MAPK,
ERK1/2 MAPK, PI3-K or PLC signaling pathway.
To elucidate the mechanism by which mycobacterial
infection affects expression of GM-CSF, we determined thesignaling pathway associated with MTB-stimulated inductionof GM-CSF. Treatment with Ro-31-8425 (an inhibitor ofcalsscial PKC) or SP600125 (an inhibitor of JNK) did not
influence of GM-CSF induction by MTB. SB202190 (aninhibitors of p38 MAPK), PD98059 (inhibitor of ERK1/2),
Ly294002 (inhibitor of PI3-K) and U73122 (inhibitor of PLC)
had negative effects on MTB-stimulated GM-CSF expression.
Mycobacterial infection induces GM-CSF secretion and theinduction of GM-CSF by MTB is mediated by ERK1/2 MAPKor PI3-K/PDK1/Akt signaling pathway.
Differentiated THP-1 cells were pre-treated with indicatedconcentrations of PD98059 (ERK1/2 inhibitor), Ly294002(PI3-K inhibitor), OSU03012 (PDK1 inhibitor) or Akt (Aktinhibitor IV) for 45 min, followed by MTB infection (10 MOI).
Supernatants were harvested 24 h after infection and secretionof GM-CSF was measured by ELISA. All signal inhibitors
were decreased GM-CSF secretion at high dose of specificsignal inhibitors. PD98059 and Ly294002 decreased about
85 % of GM-CSF secretion, OSU03012 and Akt decreased
about half of GM-CSF secretion.
Discussion
The Members of the CSF cytokine family play importantroles in macrophage recruitment and activation to regions ofinflammation. In mycobacterial infection, alveolar macrophagedisplays increasing levels of GM-CSF. However, mechanism of
GM-CSF induction does not poorly understood. We
investigated whether increasing levels of GM-CSF in
mycobacteria infected THP-1 cell mediated mycobacterial
secreted proteins. The increase in expression and secretion of
GM-CSF was caused by MTB was reduced in cells treated with
inhibitors of ERK1/2 MAPK. In addition the induction of GMCSF caused by MTB was partially reduced in cells treated with
inhibitors of p38 MAPK. In addition, the increase in secretionof GM-CSF caused by MTB was reduced in cells treated with
inhibitors of PI3-K, PDK1 and Akt. These results suggest that
GM-CSF plays important role in the immune response against
mycobacterium infection. Therefore this signal transduction
system might be a target for screeing therapeutic candidates formycobacterial disease
References[1]Szeliga J, Daniel DS, Yang CH, Sever-Chroneos Z,
Jagannath C, Chroneos ZC. Granulocyte-macrophagecolony stimulating factor-mediated innate responses intuberculosis. Tuberculosis. 2008; 88; 7–20.
[2]North RJ, Jung YJ. Immunity to tuberculosis. Annu Rev
Immunol. 2004;22;599–623.
[3]Fleetwood AJ, Cook AD, Hamilton JA. Functions of
granulocyte–macrophage colony-stimulating factor. Crit Rev
Immunol. 2005;25;405–28.
[4]Bonfield TL, Raychaudhuri B, Malur A, Abraham S,

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Trapnell BC, Kavuru MS, et al. PU.1 regulation of humanalveolar macrophage differentiation requires granulocyte–
macrophage colonystimulating factor. Am J Physiol Lung
Cell Mol Physiol. 2003;285;L1132–6.
[5]Algood, H.M., Chan, J., and Flynn, J.L., Chemokine andtuberculosis. Cytokine Growth Factor Rev. 2003;14;467–477.
[6]Bozinovski S, Jones JE, Vlahos R, Hamilton JA, Anderson
GP. Granulocyte/macrophage-colony-stimulating factor(GM-CSF)
regulateslunginnateimmunitytolipopolysaccharide through Akt/Erk activation of NF-kappa
B and AP-1 in vivo. J Biol Chem. 2002;277(45):42808-14.
Author address

E-Mail: hyelee@yonsei.ac.kr

MOLECULAR BASIS OF DECREASE OF PROTEIN S IN PREGNANCY:
17β-ESTRADIOL DOWN-REGULATES PROTEIN S (PS) EXPRESSIONRECRUITING CO-REPRESSOR COMPLEX TO THE PS PROMOTER
A. Suzuki1), Y. Miyawaki1), J. Fujita1), A. Maki1), Y. Fujimori1), A. Takagi1, 2), T. Murate1, 2),
H. Saito3) and T. Kojima1, 2)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya,Japan2) Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan
3) Nagoya Central Hospital, Nagoya, Japan

Introduction
Anticoagulant Protein S (PS) is a vitamin K-dependent
plasma protein that functions as a nonenzymatic cofactor for
activated protein C in the anticoagulation pathway [1].
Decreased levels of plasma PS are known as a risk factor forthe development of deep venous thrombosis, and there aremany factors to cause the PS deficiency [2-4].
Hereditary PS deficiency is determined by detection ofgenetic mutation in PSα gene (PROS1) [5]. PS deficiency
can also occur throughout life under the acquired conditions
such as oral anticoagulant (i.e. warfalin) use and liver disease[6]. Furthermore, acquired PS deficiency has been reported
in individuals with high levels of estrogen during pregnancy
and in those taking oral contraceptives [7-9].
Estrogens are reported as the regulator of the specific gene
expression via estrogen receptors [10]. The estrogen receptor(ER) is one of the steroid/nuclear receptor, and the ligandbound ER functions as a transcriptional factor [11]. The
classical ER action involves ligand-induced formation of an
ER homodimer, which interacts with estrogen responsive
elements (EREs) in target gene promoters [11]. The ligandoccupied ER is also known to associate with othertranscription factors, not mediating EREs to modulate liganddependent gene expression [12-15].
In this study, we investigated the molecular basis of the PS
decrease caused by E2, and thus the genomic ER signaling
pathway modulating ligand-dependent PROS1 gene
repression, in HepG2-ERα cells stably expressing human
ERα.
Materials and Methods
Establishment of HepG2-ERα cell line: HepG2 cells thatstably express human ERα (HepG2-ERα) was established byG418 selection and used for further study.
Quantitative RT-PCR and ELISA: Quantitative reverse
transcription (RT)-PCR was performed to determine PS
mRNA and GAPDH mRNA using Power SYBR Green
Master mix. Enzyme-linked immunosorbent assay (ELISA)
was carried out to determine human PS antigen.
Luciferase Reporter Assay: To determine the PROS1
promoter activity, luciferase reporter assay was performed in
respective cell lines. Luciferase activity was normalized tothe activity of co-transfected β-galactosidase.
Electrophoretic Mobility Shift Assay (EMSA), DNA Pulldown Assay and Chromatin Immunoprecipitation (ChIP)
assay: EMSA and DNA pulldown assay were carried out todetermine the transcription factors binding to the PROS1
promoterregionsusing
artificial
double-stranded
oligonucleotides. ChIP assay was performed with specificantibodies against various transcription factors boundintracellular DNA of the cells.

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Results
PS expression was repressed by 17β-estradiol in humannormal hepatocytes and HepG2-ERα cells: To determine theeffect of 17β-estradiol (E2) to the PROS1 expression, we
analyzed PS mRNA in human normal hepatocytes (hNHeps)and HepG2 or its-derived cell lines. In ERα-positive hNHeps
treated with 100nM E2, PS mRNA was decreased comparedwith vehicle-treated cells. In HepG2-ERα cells, PS mRNA andantigen were also repressed by E2 treatment, but not in ERαnegative other cell lines.
Down-regulation of PROS1 promoter activity by E2: To
investigate of the effects on PROS1 promoter activity, wecloned the promoter region of PROS1 and constructedluciferase-reporter vectors. In the full-length promoter construct(PS-4229/pGL3) and its-truncated constructs (PS-1826/pGL3 to
PS-175/pGL3), luciferase activities were down-regulated by E2,
but PS-137/pGL3 was not. There are two adjacent GC-richmotifs between -175 and -137, and PS-175Mut/pGL3
containing mutations of these GC-rich motifs did not show E2dependent repression.
ERα mediated E2-dependent PROS1 down-regulation: We
also examined the necessity of ERα expression for the PROS1
repression by E2 in HepG2-derived cells. In HepG2-mocktransfected cells, the PROS1 promoter activity was not downregulated by E2 treatment, but was in HepG2-ERα cells. A pureER antagonist, ICI 182,780, blocked this E2-induced PROS1
repression.
Transcription factor Sp1, Sp3 and ERα bound to the PROS1
promoter region, and Sp1 was necessary for E2-dependent
repression: Next, we performed the DNA pulldown assay andthe electrophoretic mobility shift assay (EMSA) to identify thetranscriptional factors interacted with the PROS1 promoter. In
DNA pulldown analyses, we found that Sp1, Sp3 and ERα
bound to the oligonucleotide containing the GC-rich motifs of
PROS1 promoter. We also observed the bindings of Sp1 andSp3 to the same oligonucleotide in EMSA, but could notdetected ERα binding. To verify the functions of Sp proteins in
the E2-induced PROS1 repression, we carried out knockdown
experiments for Sp1 and Sp3 by RNA interference using
specific siRNAs against them. The knockdown of Sp proteins
resulted in reduced promoter activities, but E2-dependent
repression did not occur by knockdown of Sp1, whereas
remaining E2-responsiveness by knockdown of Sp3.
Sp1/ERα interacted receptor interacting protein 140 (RIP140),
and recruited co-repressor-HDAC complex: We performed thechromatin immunoprecipitation (ChIP) assay and ChIP reimmunoprecipitation (IP) assay to investigate the chromatinstates of E2-treated HepG2-ERα cells. In ChIP assays, weobserved the bindings of Sp1, Sp3 and ERα to the PROS1
promoter region containing GC-rich motifs as well as DNA
pulldown assay. Sp1 and Sp3 were bound to the GC-rich motifs

in an E2-independent manner, but Sp1 binding was enhancedby E2 treatment. The binding of ERα was completely in an
E2-dependent manner. In further analyses of transcription
factors interacting with the PROS1 promoter, we found thatco-repressor nuclear receptor co-repressor (NCoR) andsilencing mediator of retinoid and thyroid hormone receptors(SMRT) were E2-dependently recruited to the GC-richmotifs. These co-repressors were not known to be recruitedby liganded ERα, however, we tried ChIP assays for receptor
interacting protein 140 (RIP140) [16] and ligand-dependent
co-repressor (LCoR) [17], which are known to interact
liganded ERα, and we identified only RIP140 bound to the
PROS1 promoter containing GC-rich motifs. Because
RIP140 is also known to interact histone deacetylases(HDACs) [18], we investigated the recruitment of them. We
observed class I HDAC; HDAC3 was robustly recruited by
E2-treatment, whereas class II HDACs did not affected.
To confirm the interaction of these transcription factors
upon the PROS1 promoter region containing GC-rich motifs,
we performed ChIP re-IP assays. The results suggested thatSp1 interacted ERα, and Sp1/ERα recruited NCoR/SMRTHDAC3 complex via RIP140. A HDAC inhibitor trichostatin
A (TSA) blocked E2-dependent repression, so PROS1 gene
repression was mediated HDACs, which induced a hypoacetylated chromatin state and less permissive transcription.
Discussion
The decreased level of plasma PS during pregnancy is awell-known risk factor for deep venous thrombosis, but its
precise mechanism has not been unknown. In this study, weclarified that PS decrease was induced by 17β-estradiol
through transcriptional repression.
Meanwhile, the action of estrogen has been investigatedwell, which is ligand-dependent modulation of estrogen
receptor and regulation of transcription in target gene.
Although the positive regulation of target gene expression by
estrogen receptor has been reported in various studies,
negative regulation of target gene expression has been lessunderstood. Especially, liganded ER had been thought toonly regulate the transcription positively. We identified
liganded ER has a potential of negative regulation, mediating
RIP140 and co-repressor-HDAC complex. RIP140 is known
to interact HDACs, whereas HDACs exist sometimes ascomplex such as NCoR/SMRT-HDAC, so there might beanother formation accompanied with ER-RIP140 upon thetarget gene promoter.
In the down-regulation of protein S gene expression, theinteraction of Sp1 and ERα might be important. The Sp1formed complex with ERα upon the GC-rich motifs of the
PROS1 promoter, and consequently they reinforced thestability of Sp1/ERα-DNA complex. The interacted ERα
recruited RIP140, and RIP140 recruited the NCoR/SMRTHDAC3 complex. The NCoR/SMRT-HDAC3 complex
might induce the hypoacetylated histones, in which DNA
could coil tightly and limited accessibility of the basaltranscription factors and thus repressing the PROS1
expression.
Conclusions
In this study, we have demonstrated that 17β-estradiol
down-regulates protein S gene expression via ERα. Liganded
ERα interacts basal transcriptional factor Sp1 and recruits
RIP140, which associates directly with HDAC3 complex
containing NCoR and SMRT. Thus E2-dependent PROS1
repression results in decreased levels of plasma PS, and thisMT-3

leads to the risk of deep venous thrombosis during pregnancy ororal contraceptive use.
References[1] Dahlbäck, B. (1991) Thromb Haemost 66, 49-61[2] Schwarz, H. et al, (1984) Blood 64, 1297-1300[3] Engesser, L. et al. (1987) Ann Intern Med. 106, 677-682[4] Makris, M. et al. (2000) Blood 95, 1935-1941[5] Gandrille, S. et al. (2000) Thromb Haemost. 84, 918[6] D'Angelo, A. (1988) J Clin Invest. 81, 1445-1454[7] Comp, P. et al. (1986) Blood 68, 881-885[8] Malm, J. et al. (1988) B J Haematol 68, 437-443[9] Tans, G., et al. (2000) Thromb Haemost 84, 15-21[10] Hall, J. et al. (2001) J. Biol. Chem. 276, 36869-36872[11] Gruber, C. et al. (2002) N Engl J Med 346, 340-352[12] Delfino, F. et al. (1999) Mol Cell Endocrinol 157, 1-9[13] Kushner, P. J. et al. (2000) J Ster Biochem Mol Biol 74,
311-317[14] Levin, C. (2005) Eur J Nuclear Med Mol Imaging 32,
S325-S345[15] Safe, S. et al. (2004) Nuclear Receptor-MediatedTransactivation Through Interaction with Sp Proteins. in
Progress in Nucleic Acid Research and Molecular
Biology, Academic Press. pp 1-36
[16] de Wolf, C. J. et al. (2006) J Biol Chem 281, 1763517643[17] Safe, S., and Kim, K. (2008) J Mol Endocrinol 41, 263275[18] Owen, G. et al. (1998) J Biol Chem. 273, 10696-10701
Author address

E-Mail: suzuki.atsuo@d.mbox.nagoya-u.ac.jp

COMPARISON OF REBA HPV-IDⓇ WITH DNA CHIP BASED ASSAY FOR HPV
GENOTYPING
Sung-Hyun Kim1)*, Dong-Sup Lee2)*, Sang-Jung Park1), Tae-Ue Kim1),
Gwang-Hwa Park2), and Hye-Young Lee1)
1) Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University2) Department of Pathology, College of Medicine, Yonsei University Wonju Christian Hospital,
Wonju-si, Gangwon-do, Republic of Korea

Introduction
Human papillomaviruses(HPV)have been known to cause
human cervical cancer which is the second most common
cancer among women worldwide and the most common cancer
among wo men in developing countries [1, 6, 9].
Even though, the HPV prevalence among subgroups ofpeople classified by disease severity varies, overall prevalenceof HPV in people with cervical cancer has been reported toreach up to 95 to 100% [9]. So far, more than 100 genotypes of
HPV have been known. Among those, certain groups of HPV
have been implicated to have high association with cervicalcancer, whereas other types have low association [2]. In detail,the most associated geno-types with malignant cervical cancer
are 16, 18, 45, 31, 33, 52, 58, and 35. Of these, HPV16 and 18
are mostly related to the malignant cervical cancer [1].
For this reason, HPV genotyping rather than mere detectionof HPV has been recommended and thus, the test volume for
HPV genotyping has been rapidly increasing. Nowadays,
reliable and accurate detection method that able to identify the
specific HPV genotypes is required, thus several PCR-based
assays have been developed to detect specific genotypes of
HPV [8]. Currently, many genotyping tests kits in clinicalsettings have been commercially available and evaluated to bevaluable for clinical diagnosis [3, 8].
This study was set to evaluate a new HPV genotyping kit,
REBA HPV-IDⓇfrom M&D (Wonju, Korea) for its usefulness
for genotyping HPV using clinical specimens.

recommendation. Briefly, for REBA HPV-IDⓇ assay, PCR
products added to the genotype specific probe labeled
membrane strips, washed non-specific binds, and then stainedwith staining solution of PCR product-probe linked region of
strips. For MyHPV DNA Chip assay, PCR product loaded tothe probe labeled glass chip and then read the result signal
using by scanner.

Materials and Methods
HPV samples and liquid based cytological smears
The clinical specimens were obtained since November of2008 to July of 2009 using by standard procedure from 356
women in Gangwon province, Korea from Yonsei university
Wonju Christian Hospital.

Genotyping of HPV in abnormal cytology, REBA HPV-IDⓇ
versus MyHPV DNA chip
The overall positivity of REBA HPV-IDⓇ was 80.86%(186) using clinical specimens with abnormal cytologicalinterpretation: the positivity rate of HPV with abnormal
cytology was 91.67% (11) in SCC, 96.66% (29) in HSIL, 92%(46) in LSIL, 100% (2) in ASC-H, and 64.17% (43) in ASCUS.
When the results from REBA HPV-IDⓇ were compared with
MyHPV DNA Chip, the overall positivity rate of MyHPVDNA chip was 69.75%, and the positivity rate of HPV withabnormal cytology was 91.67% (11) in SCC, 96.88% (29) in
HSIL, 84 % (42) in LSIL, 100% (2) in ASC-H, and 43.28%(29) in ASCUS with MyHPV DNA chip. The positive rate of
HPV in ASCUS was significantly lower with MyHPV DNA
chip than with REBA HPV-IDⓇ. These data shows that REBA
HPV-IDⓇ has about 30% higher sensitivity than MyHPV DNA
chip.
The five most frequently detected HPV sub-types using
REBA HPV-IDⓇ were HPV16, 53, 58, 56, and 33 withabnormal cytological diagnosis, while using Mygene DNA chip,

Cytological diagnosis
The Liquid-based cytological smears obtained using
recommended procedures and clinical diagnoses wereinterpreted by the pathologists and were classified by 2001
Bethesda System terminology.
HPV genotyping
HPV genotyping using REBA HPV-IDⓇ (M&D, Wonju,
Korea) and MyHPV DNA Chip (Mygene, Seoul, Korea) werecarried out according to the manufacturer's recommendation.
Both genotyping methods, REBA HPV-IDⓇ and MyHPV DNA
Chip require the target region (firstly, amplify MY11 and MY9then, amplify GP5 and GP6) amplification before performing
the examination. After amplification of target region by PCR,
post PCR steps performed according with manufacturer’s

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Sequencing
Total eight samples were analyzed by sequencing forconformational test. These eight samples have got positive
result which appear single HPV genotype infection with REBA
HPV-IDⓇ, however the result was negative with MyHPV DNA
Chip. For sequencing analysis, one-tube nested PCR was
performed to amplify the target region (GP5 and GP6) and then,sequencing analysis was performed at Xenotech (Dae-joen,
Korea).
Results
Clinical diagnosis of HPV
According to the cytological interpretations, 180 specimens(50.56%) were normal, 162 specimens (45.5%) were abnormal,
and 14 specimens (3.93%) have not been diagnosed because
which were prescribed to perform HPV genotyping directly
without cytological diagnoses. Abnormal cytological diagnoses
were classified 6 grades. Respectively, 67 (18.82%) ASCUS, 2(0.56%) ASC-H, 50 (14.04%) LSIL, 30 (8.42%) HSIL, 12(3.37%) SCC, and 1 (0.28%) CⅢB2C.

HPV subtypes 16, 58, 56, 53, and 18 were most frequently
detected.
Genotyping of HPV in normal cytology, REBA HPV-IDⓇ versus
MyHPV DNA chipOn the other hand, the rate of HPV positive was 64.44%(116) in REBA HPV-IDⓇ and, 34.44% (62) in MyHPV DNA
chip based assay with normal cytology. The data shows REBA
HPV-IDⓇ has about 2 fold higher positivity than DNA chipwith normal cytological diagnosis. In this study, The five mostremarkable HPV sub-types detected were HPV16, 53, 58, 18,
and 56 with normal cytological diagnosis using REBA HPVIDⓇ and HPV16, 52, 18, 53, and 58 using MyHPV DNA chip.
These data suggest that REBA HPV-IDⓇ has higher specificity
than MyHPV DNA chip.
Sequencing analysis with specimens that have different results
To confirm the accuracy and reliability of two methods,
sequencing analysis was performed. Before the sequencing
analysis, sorting of suitable specimens was required. Total
seven specimens, which have negative results with MyHPVDNA Chip, have a strong signal of PCR and have single
genotype infection result (one-band) with REBA HPV-IDⓇ,
were selected. GP5, GP6 region was amplified and sequenced.
Five out of seven samples sequenced clearly to interpret data
and these results were corrected with REBA HPV-IDⓇ
because these samples had negative results with MyHPV DNA
Chip. The rest two samples could not be interpreted bysequencing analysis. They would be mixed infection which
infected with more than two genotypes of HPV or non-HPV
infection (negative). Therefore, these data suggest that REBA
HPV-IDⓇ has higher sensitivity (72.42%) and accuracy than
MyHPV DNA Chip (28.57%).
Discussion
In this study, we evaluated the performance of the REBA
HPV-IDⓇ system which based on reverse blot hybridization
assay compare with the results of DNA chip assay andcytological interpretation with clinical specimens.
Recently available HPV genotyping kit REBA HPV-IDⓇ
has several advantages over a DNA chip based HPV
genotyping method. First of all, REBA does not require
expensive equipments such as scanner. Second, it is moresuitable for the laboratory with large volume of tests by saving
labor and reducing turn-around time. The total time required
for the REBA HPV-IDⓇ was from PCR to genotyping analysis
was about 4 hours. In brief, it can be more economical and time
consuming.
According to our finding, the positivity of the REBA HPVIDⓇ was higher (80.86%) than other DNA chip based method(69.75%) in abnormal cytological samples and also positivityof REBA HPV-IDⓇ (64.44%)was higher than MyHPV DNA
chip (34.44%) in normal cytological samples. Thus, it seems
that REBA HPV-IDⓇ would provide more sensitive andspecific detection rate for samples with abnormal and normal
cytology.
To confirm the reliability of REBA HPV-IDⓇ, Sequencing
analysis was performed. Sequencing results show that REBA
HPV-IDⓇ was more reliable and higher sensitivity.

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Conclusions
Sequencing analysis has a limitation that is not a golden
standard method as a confirmative test because if some patients
who infected with more than 2 HPV genotypes, the results
could not be interpreted clearly. Therefore, other kinds ofconfirmative tests are required for accurate HPV genotype
detection and need to further evaluation.
References[1] Bosch F X, Lorincz A, Munoz N, Meijer C J and Shah K,
The causal relation between humanpapillomavirus andcervical cancer. J. Clin. Pathol. 2002; 55 244–265.
[2] De Villiers EM, Fauquet C, Broker TR, Bernard HU, Zur
Hausen H, Classification of papilloma-viruses. Virology
2004; 324:17-27.
[3] Fatima Galan-Sanchez, M A Rodriguez-Iglesias,
Comparison of human papillomavirus genotyping using
commercial assays based on PCR and reverse hybridization
methods. APMIS 2009; 117: 708–15.
[4] F X Bosch, A Lorincz, N Muñoz, C J L M Meijer, K V
Shah, The causal relation between human papillomavirus
and cervical cancer. J. Clin. Pathol. 2004; 55:244–265.
[5] Roden R, Wu T C, How will HPV vaccines affect cervicalcancer? Nat. Rev. Cancer. 2006; 6:753–763.
[6] Schiffman MH, Bauer HM, Hoover RN, Glass AG, Cadell
DM, Rush BB(1993) Epidemiologic evidence showing thathuman papillomavirus infection causes most cervicalintraepithelial neoplasia. J. Natl. Cancer. Inst. 1993; 85:958–
64.
[7] S R Hong, I S Kim, D W Kim, M J Kim, A R Kim, Y O
Kim, H S Kim, Prevalence and genotype distribution of
cervical human papillomavirus DNA in Korean women : amulticenter study. Kor. J. Pathol. 2009; 43:342-50.
[8] Thomas Iftner, Liesje Germ, Ryan Swoyer, Development
and clinical evaluation of a highly sensitive PCR-reverse
hybridization line probe assay for detection andidentification of anogenital human papillomavirus. J. Clin.
Microbiol.1999;37:2508–2517.
[9] Walboomers, J. M. M., M. V. Jacobs, M. M. Manos, F. X.
Bosch, A. Kummer, K. V. Shah, and P. J. F. Snijders.
Human papillomavirus is a necessary cause of invasive
cervical cancer worldwide. J. Pathol. 1999; 189:12–19.

Corresponding Author address

E-Mail: Hyelee@yonsei.ac.kr

Morphometric and FISH Analyses of Adenocarcinoma andMesothelial Cells Using Liquid-Based Cytology
A.Morimoto1), T.Nagasaka1), 2), K.Hashimoto1), 2), T.Yokoi3)1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine,
Nagoya, Japan
2) Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan
3) Department of Diagnostic Pathology, Aichi Medical University Hospital, Nagakute, Aichi, Japan
Introduction
Cytological diagnosis of malignant effusions is often
difficult mainly because of the presence of reactive
mesothelial cells. The evaluation of cellular morphology
with conventional Papanicolaou or Giemsa stains tend to be
more or less subjective as shown by the interobserver andeven the intraobserver variabilities. Immunocytochemistry
has been applied to aid the differentiation of adenocarcinoma
and reactive mesothelial cells, but is not very useful largely
because of the limitation of the specimen. LBC is superior toconventional cytology in that it provides smear slides with auniform cell density and a distribution that enables a precise
cytological examination. Moreover, one can preserve cells in
the LBC solution for a long period of time and always utilize
it for a variety of purposes, such as immunocytochemistry
and molecular analysis. To establish a more objective
diagnostic system, we performed morphometric and FISH
analyses on adenocarcinoma and mesothelial cells, using aliquid-based cytology method, and evaluated its usefulness.
Materials and Methods
Fourty five tumors of alimentary tract, 20 gastricadenocarcinomas and 25 colonic adenocarcinomas, werestudied. Cells were harvested from the surface of tumor andserosa of resected fresh specimen, suspended in the solution
for fixation and preservation (Liqui-PREPTM Preservative
Solution), and processed for Papanicolaou staining and FISH.
Digital images were taken from Papanicolaou-stained slides
for the following measurements. The nuclear area andperimeter of 100 cells per each case were measured using thesoftware ImageJ (NIH), and the mean nuclear area (NA), themean nuclear perimeter (NP), the mean length of nuclear
longer axis (LLA) and their standard deviations (SNA, SNP,
SLLA) were calculated. The shape factor (SHF) was
obtained by the following formula: (4×π×area)/perimeter2.
SHF is dimensionless and its value is equal to 1.0 in circlesand <1.0 in spheroid nuclei (Figure 1). Standard deviationsof the shape factor (SSHF) were also calculated. FISH
analysis was performed to detect chromosome 8 polysomy
and c-myc amplification.
Results
The nuclear area (NA) was smaller in gastricadenocarcinoma cells than in colonic adenocarcinoma cells
and mesothelial cells, although the differences were notstatistically significant. Results of the standard deviation of
nuclear area (SNA) demonstrated that adenocarcinoma cells
had more variable-sized nuclei than mesothelial cells(**P<0.01) (Figure 2).
Results of the nuclear perimeter (NP) showed a similartendency to NA. As for the standard deviation of nuclear
perimeter (SNP), colonic adenocarcinoma cells showed thehighest value, followed by gastric adenocarcinoma cells, andmesothelial cells showed the lowest value (**P<0.01)(Figure 2).

MT-5

As for the length of nuclear longer axis (LLA), colonicadenocarcinoma cells had longer nuclei (*P<0.05, **P<0.01)
than gastric adenocarcinoma cells or mesothelial cells without asignificant difference between the latter two. Results of thestandard deviation of length of nuclear longer axis (SLLA)
showed a similar tendency to SNP (**P<0.01) (Figure 2).
As for the shape factor (SHF), mesothelial cells had themost rounded nuclei, followed by gastric adenocarcinoma cells,
and nuclei of colonic adenocarcinoma cells were the least
rounded (**P<0.01). Results of the standard deviation of shapefactor (SSHF) showed a similar tendency to SNP and SLLA(**P<0.01) (Figure 2).
Adenocarcinoma cells of both stomach and colon in allcases showed significant percentages of chromosome 8polysomy and c-myc amplification, whereas mesothelial cells inall cases showed normal cell pattern.
Discussion
Morphometricanalysis
revealed
thatgastric
adenocarcinoma cells were less atypical and more uniform thancolonic adenocarcinoma cells and had even smaller nuclei thanmesothelial cells. One should always take these facts into
consideration on cytological examination, since gastric andcolonic adenocarcinoma cells are definitely the most frequently
encountered atypical cells in intraoperative peritoneal washingcytology, although cellular atypia should be more integratedly
assessed using other indices. By FISH analysis using LBC
specimens, quantitation of positive signals was more easily
performed than with histology sections, resulting in detection ofchromosome 8 polysomy and c-myc amplification in significant
percentages of both gastric and colonic adenocarcinoma cells,
whereas all the mesothelial cells showed the normal pattern.
Conclusions
Morphometric and FISH analyses, together withconventional stains and immunocytochemistry, using LBC
method can be used as ancillary techniques in differentiation of
adenocarcinoma and mesothelial cells.
Acknowledgment
The author is grateful to Dr. Akihiro Ito, Toki General
Hospital, for providing clinical materials and Prof. Tsutomu
Kawabe, Nagoya University School of Health Sciences, forreviewing the manuscript and giving useful advice.
References[1]Bernstein SJ, Sanchez-Ramos L, Ndubisi B. Liquid-based
cervical cytologic smear study and conventional
Papanicolaou smears: a metaanalysis of prospective studies
comparing cytologic diagnosis and sample adequacy. Am J
Obstet Gynecol 2001;185:308-17.
[2]Nasuti JF, Tam D, Gupta PK. Diagnostic value of liquidbased (Thinprep) preparations in nongynecologic cases.
Diagn Cytopathol 2001;24:137-41.
[3]Ikeguchi M, Oka S, Saito H, Kondo A, Tsujitani S, Maeta M,
Kaibara N. Computerized nuclear morphometry: a newmorphologic
assessmentfor
advanced
gastric
adenocarcinoma. Ann Surg 1999;229:55-61.
[4]Susumu N, Aoki D, Noda T, Nagashima Y, Hirao T,
Tamada Y, Banno K, Suzuki A, Suzuki N, Tsuda H,
Inazawa J, Nozawa S. Diagnostic clinical application of
two-color fluorescence in situ hybridization that detects
chromosome 1 and 17 alterations to direct touch smear andliquid-based thin-layer cytologic preparations ofendometrial cancers. Int J Gynecol Cancer 2005;15:70-80.
[5]Koide K, Sakakura C, Hagiwara A, Yamaguchi T,
Yamagishi H, Abe T, Inazawa J. An improved rapid
procedure for fluorescence in situ hybridization that is
applicable to intraoperative cancer cytodiagnosis. Cancer
Lett 2000;158:165-9.

Figure 1. A: The shape factor (SHF) is dimensionless and its
value is equal to 1.0 in circles and <1.0 in spheroid nuclei.
B: The shape factor (SHF) of this picture shows 0.743, thenuclear area (NA) is 84.065μm2, the nuclear perimeter (NP)is 37.700μm, and the length of nuclear longer axis (LLA) is
14.183μm.

Figure 2. Results of morphometric analysis of mesothelial cells
and adenocarcinoma cells on LBC slides. MC: mesothelial cells,
GAC: gastric adenocarcinoma cells, CAC: colonicadenocarcinoma cells.
Author address

E-Mail: morimoto.ayumi@b.mbox.nagoya-u.ac.jp

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MOLECULAR MECHANISM OF CERAMIDE KINASE GENE REPRESSIONON ATRA-INDUCED NEURONAL DIFFERENTIATION
Masashi Murakami1), 2), Hiromi Ito2), Kazumi Hagiwara2), 3), Noriko Sasaki2),
Misa Kobayashi2), Asuka Hoshikawa2), Akira Takagi2), Tetsuhito Kojima2),
Keiko Tamiya-Koizumi4), Yoshiko Banno5), Yoshinori Nozawa6), Takashi Murate2), 7)
1) Research Fellow of the Japan Society for the Promotion of Science, 2) Department of Pathophysiological Laboratory
Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3) National Hospital Organization, Nagoya
Medical Center, Nagoya, Japan, 4) Division of Molecular Pathology, Aichi Cancer Center, Nagoya, Japan, 5)
Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan, 6) Gifu International Instituteof Biotechnology, Kakamigahara, Japan, 7) Department of Medical Technology, Nagoya University School of Health
Sciences, Nagoya, Japan
Introduction
Ceramide kinase (CERK) is the enzyme that
phosphorylated ceramide to ceramide 1-phosphate (C1P).
Previous studies suggested that ceramide kinase (CERK)
and its product, ceramide 1-phosphate (C1P) have been
implicated in various cellular function including cellular
proliferation, survival, mast cell degranulation, andphagocytosis.
However, the regulatory mechanism of CERK gene
expression remains to be determined.
In the current study, we examined the regulatory
mechanism of CERK gene expression during all-transretinoic acid (ATRA)-induced neuronal differentiation of ahuman neuroblastoma cell line, SH-SY5Y cells.
Materials and Methods
Cell line: A human neuroblastoma cell line, SH-SY5Y,
was cultured in Dulbecco’s Eagle’s medium supplemented
with 10% fetal bovine serum.
Establishment of CERK stable transfectants: Human
CERK cDNA was the kind gift of Dr. Kohama T. (DaiichiSankyo Co., Ltd., Tokyo, Japan). hCERK cDNA wasinserted into pcDNA3.1(+) expression vector (Invitrogen,
California, USA). To establish the stable transfectants of
CERK, DNA transfection was performed by the calcium
precipitation method. A subclone (sc 17) showing the highest
CERK protein expression was used for further experiments.
Cell proliferation and cell death: Cell proliferation wasdetermined in triplicate with WST-1 assay kit (Roche
Applied Science, Mannheim, Germany). Cell death was
determined with LDH-cytotoxic test (Wako Pure Chemical
Industry, Osaka, Japan).
CERK enzyme activity: CERK activity was determined as
described previously [1] with some modification.
Western blotting: Western blotting was performed using
ECL plus western blotting system (Amersham Pharmacia
Biotech, Buckinghamshire, UK).
Quantitative RT-PCR: Quantitative RT-PCR was
performed as described previously [2]. The primers were as
follows: CERK sense 5’-AGTCCACCACAACAGCAC-3’,
antisense
5’-GAGGAAGGTCTTTAAACCTG-3’,
Sphingosine
kinase1(SPHK1)sense
5’TCCTGGCACTGCTGCACTC-3’,
antisense
5’TAACCATCAATTCCCCATCCAC-3’, and GAPDH sense5’-CAGGAGCGAGATCCCTCCAA-3’,
antisense
5’CCCCCTGCAAATGAGCCC-3’.
Cloning of the 5’-promoter region of human CERK:
Human CERK 5’-promoter region was prepared using a
PCR-based method. Using suitable restriction enzymes, thispromoter region was inserted into pGL3 Basic vector(Promega, Madison, USA). The deletion mutants were

MT-6

prepared using Exonuclease III digestion, PCR-based cloning,
and restriction enzymes digestion method. To introduce themutation to the RARE like motif, overlap extension PCR
method was performed.
Luciferase assay: SH-SY5Y cells were plated at a density of
5x105cells/ml in 35mm dishes. After 2 days of incubation,
0.5mg of promoter construct and 2mg of b-galactosidase
expression vector were co-transfected into cells using lipofectin
reagent (Invitrogen) according to the manufacturer’s protocol.
Luciferase activity and b-galactosidase activity were measured
according to the method described previously [3].
Electrophoresis mobility shift assay (EMSA): EMSA was
performed as described previously [4]. One mg of nuclear
extract and 600 fmol of biotin-labeled probe were incubated atroom temperature for 40 min. Cold probe was used for thecompetition experiment (x20 excess). For the supershift
experiments, anti-RARa antibody, anti-RXRa antibody, andanti-COUP-TFI antibody (Santa Cruz) were used.
DNA pull-down assay: Nuclear extract, biotin-labeled
double strand probe and Poly dI-dC were incubated in DNAP
buffer for 30min on ice. Following the incubation, Dynabeads
M-280 streptavidin (Invitrogen Dynal AS, Oslo, Norway) wereadded and mixed with rotator at 4oC for 30 min. Prior to thisstep, the beads were washed in DNAP buffer for 10 min withrotator at three times. After washing, dissolved in SDS sample
buffer and heated at 95oC for 5 min. Western blotting was
performed using anti-RARa, anti-RXRa, and anti-COUP-TFI
antibody.
Chromatin immunoprecipitation assay (ChIP assay): ChIP
assay was performed as described previously [4]. For
immunoprecipitation, normal IgG, anti-RARa, anti-RXRa,
anti-COUP-TFI, anti-N-CoR, anti-SMRT, and anti-HDAC3
antibody (Santa Cruz) were added to the lysate. The CERK
promoter region was amplified by PCR using the following
primerset.
Sense:
5’-GTCCCCTCCGCGGTCCCC-3’,
antisense: 5’-GCTTCACCCACAGCACGGATT-3’.
Transient transfection followed by co-immunoprecipitation:
The plasmids of RARa, RXRa, and COUP-TFI were the giftof Dr. W. H. Miller (McGill University, Quebec, Canada), Dr.
R.M. Evans (The Salk Institute for Biological Studies, CA,USA), and Dr. G. Salbert (Universite de Rennes I, Rennes,
France), respectively. After transfection with respective plasmid
DNA using lipofectin reagent (Invitrogen), SH-SY5Y cells weretreated with either ATRA or methoprene acid, a specific agonistof RXRa, for 24 h. Co-Immunoprecipitation assay was
performed by described previously [5] with some modification.
Results
ATRA reduced CERK enzyme activity, CERK protein, andmRNA expression whereas another sphingolipid metabolic

enzyme, SPHK1 that had similar function to CERK, did notshow significant changes with ATRA.
To examine transcriptional regulation of ATRA-induced
CERK gene repression, promoter analysis was performed.
ATRA reduced CERK promoter activity, and thisreduction was mainly because of the region between -40 and-28 bp from the first exon. In this region, the tandem retinoic
acid responsive element (RARE) like motifs were identified
with one base space. Introduction of mutation into these
RARE like motifs, erased ATRA-induced inhibition of
CERK promoter activity.
To determine which transcription factor was bound inthese RARE like motifs, EMSA, DNA-pull down assay, andChIP assay were performed.
These experiments revealed that ATRA induced bindingof retinoic acid receptor a (RARa), retionid X receptor a(RXRa),
Chickenovalbumin
upstreampromotertranscription factor I (COUP-TFI) to these RARE like motifs.
Moreover, ChIP assay showed that HDAC3, N-CoR, and
SMRT were recruited to these RARE like motifs.
Transient expression of RARa, RXRa, and COUP-TFI
and siRNA transfection of these genes concluded that
COUP-TFI inhibited CERK mRNA.
Another ATRA-induced differentiation model, humanacute myeloblastic leukemia cell line, HL60 cells, showedsome increase of CERK mRNA with ATRA. Interestingly,
ATRA induced binding of RARa and RXRa to these RARE
like motifs, but COUP-TFI didn’t.
These results suggested that the relative ratio of
RAR/RXR and COUP-TFI/RXR or COUP-TFI/RAR might
determine the level of CERK transcription in different celllines.
To examine a function of CERK on cell proliferation,
cell death, and neuronal differentiation of SH-SY5Y cells,
we established stable transfectants of CERK. CERK overexpression counteracted ATRA-induced inhibition of cellproliferation and cell death under the serum starvation. On
the contrary, siRNA of CERK enhanced the effect of ATRA
on cell growth and cell death.
Furthermore, to examine the effect of neuronaldifferentiation, ATRA-induced neurite extension and growth
associated protein-43 (GAP-43) protein expression, which
was main component of growth cone, was examined. CERK
over-expression
inhibited
ATRA-induced
neuronaldifferentiation. On the contrary, siRNA of CERK enhanced
neuronal differentiation.
These results revealed that CERK promoted cellproliferation and cell survival, and had an inhibitory effectagainst ATRA-induced neuronal differentiation.
Discussion
Recent analysis revealed that sphingolipid and their
metabolic enzymes were involved in cellular signalling, cell
survival, and cell death. CERK and its product, C1P havealso been suggested to play an important role of cell survival
and inflammation [6, 7]. Intriguingly, CERK is mainly
distributed in brain, especially, highly expressed in
cerebellum [8].
Our results of stable transfectants of CERK and siRNAof CERK not only confirmed previous results but also
revealed CERK as the unique modulator of neuronal cellfunction in SH-SY5Y cells. It is very interesting that further
analysis of elucidating the function of CERK in central
nervous system.
We recently reported that SPHK1 gene expression wasincreased during glial-cell line derived neurotrophic factor(GDNF) induced neuronal differentiation of humanMT-6

neuroblastoma cell line, TGW cells [9]. But in ATRA-induced
neuronal differentiation of SH-SY5Y cells, SPHK1 expression
didn’t show any change. This result suggested that CERK andSPHK1 play non-overlapping roles in neuronal differentiation.
Vitamin A and its major metabolite, ATRA, are essential for
network formation during early brain development [10]. COUPTFI involvement in modulating ATRA-induced differentiation
has been reported in multiple cell types [11, 12]. Our results
suggested that COUP-TFI modulated ATRA-induced CERK
expression, and the possible role of CERK in the early
physiological process of normal neuronal differentiation.
Conclusion
CERK is antagonistic to ATRA-induced neuronaldifferentiation and that a tandem RARE like motifs located in
the 5’-promoter region was responsible for ATRA
responsiveness, and found that COUP-TFI induced with ATRA
treatment was responsible for CERK gene repression.
References[1]Bajjalieh SM, Martin TF, Floor E. Synaptic vesicle ceramide
kinase. A calcium-stimulated lipid kinase that co-purifies withbrain synaptic vesicles. J. Biol. Chem. 1989;264:14354-14360.
[2]Sobue S, Iwasaki T, Sugisaki C, et al. Quantitative RT-PCR
analysis of sphingolipid metabolic enzymes in acute leukemia
and myelodysplastic syndromes. Leukemia 2006;20:2042-2046.
[3]Nakade Y, Banno Y, T-Koizumi K, et al. Regulation of
sphingosine kinase 1 expression by protein kinase C in a humanleukemia cell line, MEG-O1. Biochim. Biophys. Acta
2003;1635:104-116.
[4]Sobue S, Hagiwara K, Banno Y, et al. Transcription factor
specificity protein 1 (Sp1) is the main regulator of nerve growth
factor-induced sphingosine kinase 1 gene expression of the rat
pheochromocytoma cell line, PC12. J. Neurochem.
2005;95:940-949.
[5]Lee HK, Park UH, Kim EJ, Um SJ. MED25 is distinct from
TRAP220/MED1 in cooperating with CBP for retinoid receptor
activation. EMBO J. 2007;26:3545-3557.
[6]Gomez-Munoz A, Kong JY, Parhar K, et al. Ceramide-1phosphate promotes cell survival through activation of thephosphatidylinositol 3-kinase/protein kinase B pathway. FEBS
Lett. 2005;579:3744-3750.
[7]Mitsutake S, Kim TJ, Inagaki Y, et al. Ceramide kinase is amediator of calcium-dependent degranulation in mast cells. J.
Biol. Chem. 2004;279:17570-17577.
[8]Mitsutake S, Yokose U, Kato M, et al. The generation andbehavioral analysis of ceramide kinase-null mice, indicating afunction in cerebellar Purkinje cells. Biochem. Biophys. Res.
Commun. 2007;363:519-524.
[9]Murakami M, Ichihara M, Sobue S, et al. RET signalinginduced SPHK1 gene expression plays a role in both GDNFinduced differentiation and MEN2-type oncogenesis. J.
Neurochem. 2007;102:1585-1594.
[10]Ross SA, McCaffery PJ, Drager UC, De Luca LM.
Retinoids in embryonal development. Physiol. Rev.
2000;80:1021-1054.
[11]Beland M, Lohnes D. Chicken ovalbumin upstream
promoter-transcription factor members repress retinoic acidinduced Cdx1 expression. J. Biol. Chem. 2005;280:1385813862.
[12]Zhuang Y, Gudas LJ. Overexpression of COUP-TFI in
murine embryonic stem cells reduces retinoic acid-associatedgrowth arrest and increases extraembryonic endoderm gene
expression. Differentiation 2008;76:760-771.
Author address

E-Mail: murakami.masashi@c.mbox.nagoya-u.ac.jp