J Nutr Sci VitaminoI, 51, 426-432,
Expression
of Calbindin-D9k,
Process
by Which
VDR
and
Cdx-2
Messenger
RNA
Increase
Calcium
Fructooligosaccharides
Absorption
Akiko FUKUSHIMA1, Atsutane
2005
in the
in Rats
OHTA2, Kensuke
SAKAI2 and Keiko SAKUMA1,*
1Department of MolecularNutrition
, Kagawa Nutrition University, 3-9-21 Chiyoda,
Sakado, Saitama 350-0288, Japan
2Department of AppliedNutrition
, Josai International University,1
Gumyo, Togane,Chiba 283-8555, Japan
(Received June 9, 2005)
Summary
We have previously shown, through evidence that the expression of calbindin
- D9k protein shows a dose-dependent change in the intestine, that calbindin-D9k plays a role
in the ability of a fructooligosaccharide (FOS)diet to increase calcium absorption. This study
shows that the regulation of calbindin-D9k expression occurs at the transcriptional level in
a segment-specific manner, decreasing in the proximal intestine and increasing in the co
lorectal segment. To determine the transcriptional regulation of the FOS diet on calbindin
- D9k expression, two transcription factors, vitamin D receptor (VDR) and cdx-2, were ana
lyzed during 10d feeding of the FOS diet. The mRNA expression of VDR and cdx-2 was influ
enced by the FOS diet and showed a segment-specific change. In the proximal small
intestine, there was a significant correlation between the changes in both mRNAs (r=0.69,
p<0.01), while the expression of calbindin-D9k correlated neither with VDR nor with cdx
2. This means that the transcriptional change induced by the FOS diet was not regulated by
VDR and cdx-2. In the colorectal segment, there were significant correlations between gene
expressions of calbindin-D9k vs. VDR, r=0.73, p<0.01 and calbindin-D9k vs. cdx-2,
r=0.52, p<0.05. These results suggested that both transcription factors, VDR and cdx-2,
were involved in the regulation of calbindin-D9k gene expression in the colorectal segment
during the process through which the FOS diet enhanced calcium absorption.
Key Words
fructooligosaccharides,
calbindin-D9k,
Since osteoporosis is one of the most serious lifestyle
related diseases, calcium has long been a target of
nutrition research. Absorptive enterocytes lining the
intestinal villi are responsible for the terminal digestion
and absorption of calcium and other nutrients. Active
calcium absorption is by way of the transcellular path,
with three primary components participating in series
in the calcium absorption process. These components
include epithelial calcium channels (CaT1), which
enhance the luminal calcium in the absorbing entero
cyte; intracellular calbindin-D9k, which binds calcium
at high affinity and diffuses calcium across the cyto
plasm of enterocytes from the brush border membrane
to the basolateral side; and the ATP-activated basolat
eral membrane calcium pump, which transports cyto
solic calcium into the extracellular fluid of the lamina
propria (1). The expression of calbindin-D9k is largely
regulated by the active form of vitamin D3, 1,25 dihy
droxyvitamin D3 (2, 3), and stimulated by calcium re
striction in the duodenal segment (3). A high correla
tion between its concentration and calcium absorption
has been observed (4); therefore, calbindin-D9k is
thought to play an important role in intestinal calcium
*To whom correspondence should be addressed .
E-mail, [email protected]
vitamin
cdx-2
absorption.
Though many diets have been recommended to
improve calcium absorption, 1,25 dihydroxyvitamin D3
is the only dietary factor that increases calcium absorp
tion; its mechanism of action has been analyzed at the
molecular level (5). Vitamin D receptor (VDR)is also a
transcription factor and its binding sequence (VDRE)on
the promoter has been determined (5). Indigestible car
bohydrates including fructooligosaccharides (FOS) (6,
7), resistant starch (8), guar gum hydrolysate (9) and
inulin (10) have been intensively studied as dietary fac
tors that can improve calcium absorption. Among
them, FOS, a mixture of indigestible and fermentable
sugars, has been well examined. Ohta et al, reported
that FOS increased calcium absorption in balance stud
ies of rats in vivo and that calbindin-D9k was involved
in this effect (11). In the colorectal segment, the degree
of enhancement of calcium absorption and the change
of calbindin-D9k expression positively correlated with
the amount of ingested FOS. Moreover, analysis of fecal
pellets in the colorectal segment confirmed that this
stimulatory effect of FOS on calcium absorption took
place in this segment (7). The molecular mechanism by
which these indigestible carbohydrates enhance cal
cium absorption remains to be clarified.
The
426
D receptor,
calbindin-D9k
gene
was
isolated
and
the
5•L-regu
Calbindin Expression by Fructooligosaccharides
latory
region
binding
was
studies
identify
a
using
can
differentiate
the
consensus
the
vitamin
is the
only
- D9k
to
express
mRNA
in
was
expression
(4)
the
sequences
ulatory
region
interaction
of
VDR
in
have
the
The
the
Several
in
been
D receptor
(VDR)
stimulation
of
level
candidate
the
experi
calbindin
transcriptional
VDR.
calbin
culture
the
(13,
and
1Fructooligosaccharides
(FOS, Meioligo-P, Meiji Seika
Kaisha, Tokyo, Japan; concentration of oligosaccharides
was >95% of total mixture).
2Prepared according to AIN-9 3 formation (19) .
consensus
calbindin-D9k
suggested
5•L reg
14),
but
their
speculative.
Caudal-related homeodomain protein cdx-2 is
another potent transcription factor that positively regu
lates calbindin-D9k expression (15, 16); its binding
sequences were identified at -38--23
(15) and
-3439--3433
(16) of the calbindin-D9k gene. Cdx-2
also mediates the transcriptional activation of the intes
tine-specific sucrase-isomaltase (17) and lactase-phlo
rizin hydrolase (18) genes.
First, we determined
whether the regulation
of calbi
ndin-D9k
gene expression
was involved in the process
by which the FOS diet enhanced
calcium absorption.
In
addition,
the mechanism
of the FOS-mediated
regula
tion of calbindin-D9k
gene expression
was explored by
taking into consideration
the transcription
factors VDR
and cdx-2.
All of the experiments
reported
here
approval from the Animal Care Advisory
Kagawa Nutrition
University.
Animals
Dawley
and
rats
vidually
(CLEA
room
12-h
dark
according
to
experimental
of
Preparation
contents
cells
of
were
were
(upper
Both
and
of
46%
nystose,
Other
dietary
tal
Yeast
Co.,
The
contained
both
(10%
diets.
Japan)
and
is
9%
components
Ltd.
Tokyo,
55%
AIN-93G
diet
Tokyo,
Inc.,
(Tokyo,
Japan)
and
relative
control
humidity)
diet
in place
diet).
FOS
(Meioligo-P,
a mixture
of
Table
of
a
purchased
DNA
Lowry
sucrose
at a
for
1 shows
the
Meiji
42%
Seika
1-kestose,
from
Orien
Japan).
Rats were divided into three groups of 12 rats each,
the two experimental
groups and one control group. All
rats were fed 15g diet/d on day 1,18g
diet/d on day 2,
and 20g diet/d on days 3-10 and were allowed free
access to water throughout
the experimental
period.
The diet dosage was designed to obtain a similar growth
rate for the experimental
and control groups without
any diet remaining
unconsumed.
The experimental
diet
group rats were sacrificed at days 5 and 10. The 12 con
trol rats were sacrificed on day 10. Six rats from each
group were used for protein and DNA determinations,
and the remaining
6 rats were used for RNA analysis.
was
fraction
for
et al.
Western
of
pH
7.4,
30min
(22),
and
blot
analysis.
and
obtained
was
the
measured
appropriate
by
protein
by
slide.
of
containing
Appropriate
subjected
determined
The
intestine
glass
volumes
KCl.
Schmidt
(21).
a
four
4.74mmol/L
method
supernatant
the
and
content
reaction
in
were
luminal
mucosal
small
with
buffer,
method
the
saline,
proximal
segment
homogenate
the
cold
homogenized
and
the
by
39,000•~g
1F-ƒÀ-fructofuranosylnystose.
were
NaCl
After
with
the
Tris-HC1
of
amine
from
were
13.7mmol/L
(20).
prepared
(19),
FOS
indi
with
was
formulation
FOS
were
humidity-con
samples.
out
colorectal
samples
extraction
Sprague
intestinal
washed
scraped
half)
amounts
male
temperature
cycle.
diet
composition
Kaisha,
a
and
the
100g/kg
Five-week-old
Japan,
in
(25•Ž
light:
level
diets.
housed
trolled
AND METHODS
received prior
Committee
of
Analysis of colorectalcontents. The pH of the colorec
tal contents was measured directly using a compact pH
electrode (B-112, Horiba, Kyoto, Japan) before scraping
the mucosal cells.
0.12mol/L
MATERIALS
diets.
D3
of
vitamin
that
(VDRE)
remains
for
with
cell
of experimental
However,
scarce.
expression
Composition
which
(2).
are
1.
cis-ele
responsible
in
the
suggests
at
action
for
its
Table
vitro
to
Caco-2,
interacts
and
that
decreased
occurs
involves
directly
vivo
fact
mice
are
In
1,25-dihydroxyvitamin
that
The
and
line
stimuli
D metabolite
13).
13).
conducted
calbindin-D9k
dietary
expression
knockout
cell
that
from
(12,
were
factor
carcinoma
nutrient
(2,
detail
analysis
sequences
active
ments
in
vivo
transcription
colon
derived
din-D9k
ex
specific
ment
signals
analyzed
and
427
to
DNA
Thannhauser
the
diphenyl
content
of
the
centrifugation
by
the
amount
at
method
was
of
used
Western blot analysis. Western blot analysis was
basically conducted as described previously by Ohta et
al. (11) with some modifications. To transfer the pro
teins that were separated by Tricine-SDS-PAGE,Immo
bilonTM-P (Millipore Corporation, Billerica, MA) was
used. After immunoreaction, immunogenic calbindin
- D9k bands were identified using a second antibody con
jugated to horseradish peroxidase followed by light
emission from the oxidation of luminol. These chemilu
minescence signals were quantified by a LAS1000plus
luminescent imaging analyzer (Fuji Photo Film Co.,
Ltd., Tokyo, Japan). The means of the six lanes from
each group on a membrane were calculated and com
pared statistically.
RNA isolation and Northern blot analysis. Total RNA
and poly(A)+ RNA were prepared using Wako ISOGEN
(Wako Pure Chemical Industries, Ltd., Osaka, Japan)
and Oligotex-dT30 super (Takara Shuzo, Kyoto, Japan),
428
FUKUSHIMA A et al.
respectively,
in
instructions.
Calbindin-D9k
(24)
were
kindly
versity
of
from
EcoRI
fragment
the
the
coding
A
cDNA
the
lanes
the
Statistical
simple
test
(26)
linear
a
blot
from
bases
published
was
used.
BAS-2000
Co.,
group
Bio
Ltd.).
was
Each
corrected
for
statistically.
were
expressed
by
Version
considered
regression
VDR,
the
compared
(SPSS
were
and
For ƒÀ-actin
Film
analyzed
by
car
comprising
using
Values
were
1,784
(1784)
cDNA
each
Uni
frag
amplified
AJ 278466).
were
the
Northern
to
Photo
analysis.
Differences
primers
from
means
Data
Tukey's
was
according
(Fuji
six
, and
and
for
cdx-2
performed
Analyzer
means•}SD.
A
for
number
was
a
Table 2. Weight of intestinal mucosa, acidity, amount
of mucosal DNA and levels of calbindin-D9k based on
protein or DNA in rats fed control or fructooligosac
charide (FOS)-containing diets.1
cDNA
EcoRI
and
probes
using
of
by
pUCVDR
full-length ƒÀ-actin
Ouantitation
IL).
270
calbindin-D9k
the
449-477
(accession
of
of
as
VDR
E DeLuca
plasmid
probe
detection,
ƒÀ
-actin
H.
pICaBP-270
used
and
sequence
value
and
A
the
279-bp
intestinal
199-227
imaging
manufacturers'
Dr.
sequences
were
analysis.
(25)
the
(23)
by
plasmid
from
respectively,
rat
provided
with
cDNA
Wisconsin-Madison.
ment
rying
accordance
as
one-way
6.0,
SPSS,
significant
equation
ANOVA
Chicago,
at
was
p<0.05.
calculated
by
1 Values
the
least-squares
method
using
Microsoft
Excel
are
common
7.0
(Microsoft,
Tokyo,
means•}SD
, n=6.
Version
superscript
a,b Values
letters
are
in
a row
with
significantly
no
different,
Japan).
p<0.05.
RESULTS
The FOS diet increased weight of intestinal mucosa, and
amount of mucosal DNA
There was no significant difference in body weight
gain during 10d feeding between rats fed the FOS diet
and rats fed the control diet (data not shown). The con
tents of the colorectal segment of the large intestine
increased in acidity after 5d feeding of the FOS diet
(p<0.001) (Table 2). The mucosal weights of the proxi
mal small intestine and the colorectal segment of rats
fed the FOS diet were 53.0% higher (p<0.001) and
20.0% higher than those of the control rats (p<0.001),
respectively. The total mucosal DNA content was also
higher than that of the control by 20.8% (p<0.005) in
the proximal small intestine segment and 41.4%
(p<0.001) in the colorectal segment.
The relative concentration of calbindin-D9k protein
was expressed on both protein and DNA bases, each
showing the same pattern of change resulting from the
FOS diet; on a protein basis, 54.8% less in the proximal
small intestine segment (p<0.001) and 239% more in
the colorectal segment (p<0.001); similarly, on a DNA
basis, 53.0% less in the proximal small intestine seg
ment (p<0.05) and 212% more in the colorectal .seg
ment (p<0.001) (Table 2).
Calbindin-D9k mRNA expression was differently regulated
by the FOS diet in the proximal small intestine and colorec
tal segment
The
pattern
Northern
FOS
diet
is
intestine
ment
an
calbindin-D9k
analysis
shown
panel
internal
are
control.
shown
Fig.
in
B. The
in
mRNA
after
in
segment
in
group
of
blot
5
1,
panel
pattern
The
means
Fig.
2 as
and
with
A
the
a percentage
of
proximal
the
of ƒÀ-actin
in
feeding
the
and
of
expression
10d
small
colorectal
is also
six
rats
of
the
from
the
as
each
control
of
the
the
values
FOS
colorectal
the
2).
on
proximal
a
263%
at
10d.
(p<0.01)
FOS
diet
by
the
colorectal
the
FOS
proximal
diet
proximal
expression
segment,
cal
(p<0.01)
and
than
258%
was
was
differently
small
intestine
in
showed
(p<0.01)
mRNA
and
small
segment
5 d
the
segment)
protein
at
in
a significant
intestine
calbindin-D9k
expression
in
the
the
colorectal
increase
Therefore,
mRNA
the
was
less
effect
feeding
for
46.3%
while
The
of
there
small
was
group,
regulated
VDR
for
5d
calbindin-D9k
mRNA
control
and
p<0.01
effect
the
only
2B),
p<0.01
In
bindin-D9k
was
(Fig.
r=0.94,
(Table
for ƒÀ-actin.
after
(r=0.96,
intestine,
with
corrected
appeared
segment
correlation
the
were
diet
expression
segment
specific.
regulated
and
by
the
colorectal
seg
ment
The
actin
patterns
(an
ing
of
the
mal
small
tal
segment
values
a
of
the
that
in
six
rats
in
by
and
VDR
differently
shown
B.
each
the
control
the
gene
in
A and
of
are
at
was
small
the
the
shown
The
5d
In
higher
10d.
regulated
intestine
and ƒÀ-
10d
feed
the
proxi
colorec
corrected
in Fig.
4 as
expression
intestine
10d.
a 102%
VDR
3, with
panel
at
at
of
5 and
means
small
(p<0.001)
proximal
Fig.
group.
(p<0.01)
(p=0.74)
after
group
proximal
expression
the
analysis
in
The
from
showed
48.0%
in
blot
mRNA
segment
48.1%
level
segment
5d
are
panel
of
control
orectal
diet
intestine
mRNA
decreased
Northern
control)
PUS
percentage
VDR
at
of
internal
and
of
segment
increased
contrast,
level
Figure
to
the
col
(p<0.001)
4 indicates
by
and
the
FOS
diet
colorectal
segments.
seg
shown
group;
Cdx-2 mRNA expression was differently regulated by the
FOS diet in the proximal small intestine and colorectal seg
ment
The patterns
of Northern
blot analysis of cdx-2 and
Calbindin Expression by Fructooligosaccharides
Fig.
Fig.
1.
Northern
mRNA
of
blot
analysis
intestinal
of
segments:
calbindin-D9k
(A)
3.
Northern
and
out
(B)
colorectal
segment
proximal
of
fructooligosaccharides,
FOS)
rats
or
small
fed
(VDR)
intes
control
10%,
FOS
mRNA
10d. Total
RNA
was
extracted
from
for
mucosal
proximal
small
intestine
or
colorectal
and
(without
5
cells
was
segment,
further
was
purified.
analyzed
on
The
an
mRNA
agarose
or
of
gel.
and
calbindin-D9k
hybridized
cDNA.
with
out
from
all
membrane,
conducted
for ƒÀ-actin
mRNA.
position
Fig.
2.
of
the
mRNA
of
intestine
(A)
and
control
(without
FOS
as
diets
the
from
colorectal
arrow
5 or
a percentage
10d.
the
(B)
of
expressed
as
common
superscript
the
values
of
means•}SD,
n=6.
mRNA
the
are
fed
10%
was
group
in
a row
and
with
significantly
control ƒÀ-actin
FOS
corrected
in
Fig.
6
are
values
of
as
sion
31.2%
recovered
to
small
less
It
by
intestine
rats
no
FOS)
was
small
was
was
a
or
10%,
extracted
FOS
from
intestine
further
or
purified.
analyzed
on
membrane,
diets
mucosal
colorectal
The
an
and
VDR
washed
cDNA
out
from
conducted
the
mRNA
and
5 or
centage
expressed
common
different.
and
each
the
(p<0.01)
FOS
after
5d
108%
A),
and
diet
differently
segment.
In
the
expres
significantly
at
10d.
a
Con
gradual
at
expression
in
the
shown
the
higher
gene
of
are
showed
cdx-2
feeding
group.
(p=0.12)
that
colorectal
group
(panel
level
10d
means
control
segment
clear
the
from
5 and
5. The
segment
expression
is
Fig.
of
control
with
after
in
colorectal
(p<0.001).
regulated
six
intestine
the
the
increase,
shown
a percentage
small
versely,
mRNA
diet
proximal
was
control
seg
mRNA
frac
agarose
gel,
hybridized
trans
with
. Thereafter,
all
(ƒ¿-3
radioactiv
the
membrane,
and
for ƒÀ-actin
rehybrid
mRNA.
The
position
of
the
VDR
and ƒÀ-actin
arrow
mRNA.
the
of
VDR
from
colorectal
the
proximal
segment
(B)
fructooligosaccharides.
10d.
of
as
Levels
the
of
FOS)
mRNA
values
of
means•}SD,
superscript
were
the
n=6.
letters
intestine
rats
or
fed
1.0%
control
FOS
calculated
control
Values
are
small
of
in
significantly
as
diets
a per
group
a row
and
with
no
different,
p<0.05.
internal
the
4.
(A)
for
p<0.05.
of
proximal
was
(without
calculated
control
Values
letters
rats
or
were
Fig.
small
of
FOS)
of
small
fed
indicates
proximal
segment
Levels
rats
mRNA.
fructooligosaccharides,
for
of
was
and ƒÀ-actin
calbindin-D9k
D receptor
proximal
rehybridization
The
calbindin-D9k
segment
RNA
mRNA
to
indicates
the
the
(1.0ƒÊg)
ization
was
Total
2P)dCTP-labeled
radioactivity
and
colorectal
(A)
to
(ƒ¿-32P)dCTP-labeled
Thereafter,
the
vitamin
fraction
transferred
ity
washed
(B)
10d.
and
ferred
a membrane,
the
and
tion
(1.0ƒÊg)
of
segments:
fructooligosaccharides,
5
ment,
mRNA
analysis
intestinal
of
cells
the
of
(with
diets
for
or
blot
(CaBP)
intestine
tine
429
the
10d
Putting all results together, there were significant
correlations in the colorectal segment between calbin
din-D9k vs. VDR, r=0.73 (p<0.01) and calbindin-D9k
vs. cdx-2, r=0.52 (p<0.05). Between the two tran
scription factors, there was a significant correlation in
the proximal small intestine (VDR vs. cdx-2, r=0.69,
p<0.01), while no correlation
orectal segment.
was evident in the col
DISCUSSION
was
proximal
As a novel tool to clarify the mechanism
of calcium
absorption,
we used the fact that FOS increases calcium
430
FUKUSHIMA A et al.
rats fed the FOS diet more than in control rats. The cor
relation between the increase of weight of mucosal cells
and DNA suggested the stimulated proliferation of intes
tinal mucosal cells. This result is consistent with our
Fig.
5.
nal
Northern
blot
segments:
orectal
(A)
segment
of
gosaccharides.
Total
imal
small
was
further
and
rats
FOS)
RNA
analyzed
analysis
was
or
or
purified.
an
hybridized
Thereafter,
with
all
membrane,
mRNA.
cdx-2
and ƒÀ-actin
FOS
diets
colorectal
for
transferred
of
or
10d.
the
prox
and
to
mRNA
was
a membrane.
cdx-2
washed
was
indicates
col
(1.0ƒÊg)
(ƒ¿-32P)dCTP-labeled
arrow
5
cells
fraction
was
intesti
(B)
fructooli
segment,
mRNA
rehybridization
The
of
and
(without
mucosal
radioactivity
and
actin
control
10%
gel,
mRNA
intestine
from
This
agarose
cdx-2
small
fed
extracted
intestine
on
of
proximal
cDNA.
out
from
conducted
the
the
for ƒÀ-
position
of
the
mRNA.
previous observation using cDNA expression array fil
ters (28). We provided evidence that the FOS diet
enhanced the expression of genes that are involved in
cell proliferation. However, our observation was incon
sistent with other evidence on the effects of butyrate.
Among several short-chain
fatty acids that were pro
duced from FOS by colonic bacteria (29), butyrate was
considered as the most likely potential factor to regulate
expression of genes in the intestine (30). Moreover,
butyrate was thought to be a direct factor in the action
of dietary fibers that protect against colon carcinogene
sis by stimulating cell differentiation and apoptosis and
inhibiting cell proliferation (30). In contrast to these
effects, butyrate is a stimulant of normal colonic cell
proliferation in vivo (31, 32), utilizing butyrate as an
energy substrate, and normal colonic mucosa in vitro.
Although the molecular mechanisms
responsible for
the effects of butyrate in colonocyte biology are not
known, butyrate
works differently against normal
colon cells and colon cancer cells.
The calbindin-D9k
protein
calculated
on the basis
of
both protein and DNA showed the same change
as a
result of the FOS diet-a
decrease in the proximal small
intestine
and an increase
in the colorectal
segment.
Moreover, there was a significant
correlation
between
changes of protein and mRNA and transcriptional
reg
ulation was suggested.
Furthermore,
these results sug
gest that the increase
from FOS was caused
port of calcium
port.
Several
rather
6.
tine
mRNA
(A)
(without
for
5 or
centage
expressed
common
and
of
cdx-2
colorectal
from
the
segment
(13)
fructooligosaccharides.
10d.
of
as
Levels
the
of
FOS)
mRNA
values
of
means•}SD.
superscript
proximal
were
letters
control
Values
are
rats
or
intes
fed
control
10%
FOS
calculated
the
n=6.
of
small
and
have
been
in
significantly
as
cdx-2,
of
cess
by
their
arc
(33-36).
factors:
Their
adjacent
Thus,
transcription
it is
gene
trans
HNF-lƒ¿,
cis-elements
binding
to
factors
calbindin-D9k
passive
corresponding
(33-36).
gene
these
tors
paracellular
transcription
and
analyzed
other
that
diets
the
tempting
are
sites
in
box
and
TATA
to
the
to
speculate
important
regula
expression.
During
the
increases
calcium
pro
a per
group
a row
each
than
intestinal
VDR,
calbindin-D9k
Fig.
of calcium absorption
resulting
by the transcellular
active trans
and
with
no
different,
p<0.05.
tion,
the
VDR
and
mRNA
prominently compared to those of control rats. As
shown in Table 2, the contents of the colorectal seg
ment increased in acidity, and the weight of mucosal
cells and mucosal DNA of both segments increased in
cdx-2
the
diet
occurred
son,
mRNA
be
Although
occurs
in
and
between
would
increase
diet
intestine
FOS
interrelationship
mostly
(37),
proximal small intestine and colorectal segment of rats
fed an FOS diet. Observation of the tissues of rats fed the
FOS diet indicated that all intestinal segments grew
the
levels.
cium
absorption and that this increasing effect takes place in
the colorectal segment (7, 27). To analyze the mecha
nism by which FOS increases calcium absorption, the
mRNA expression of calbindin-D9k and two transcrip
tion factors, VDR and cdx-2, was determined in the
which
was
of
the
as
absorption
the
in
absorption
segment
due
(7);
the
their
ingested
small
calcium
from
and
changes
of
proximal
colorectal
extracted
colorectal
calbindin-D9k
reflected
the
in
absorp
cal
intestine
to
for
proximal
the
this
FOS
rea
small
segments.
The conclusion
of our study is that calbindin-D9k
is
involved in the process by which FOS increases calcium
absorption
in the colorectal
segment. The expression
of
Calbindin-D9k
in the proximal small intestine segment
seemed to be regulated
in a different manner
from that
in the colorectal segment without the effect of VDR and
cdx-2. Although
neither factor correlated
with calbin
din-D9k
in the proximal
small intestine,
they showed
similar changes as a result of the FOS diet. The expres
Calbindin
Expression
by Fructooligosaccharides
sion profile of calbindin-D9k is specific both develop
mentally from fetus to adult (3, 38) and regionally
along the longitudinal axis of the intestine (39), with
the most abundant expression in the proximal small
intestine (37). A very precise and complicated network
of transcription factors and butyrate has been sug
gested by many lines of evidence. Cdx-2 was stimulated
by butyrate (40), and the cis-element that interacts
with cdx-2 was identified in the VDR promoter region
(41). Co-transfection of Caco-2 cells and several trans
genic mice with various combinations of transcription
factors has revealed a complex pattern of effectiveness
that differs from the sum of the activation of any of
these factors alone (39). For the same promoter, some
combination among the network decides the develop
mental and region-dependent regulation that is specific
for the genes of the intestine including calbindin-D9k
(18), sucrase-isomaltase (17) and lactase-phlorizin
hydrolase (42).
Even though
all of the transcription
factors and cis
elements were determined,
complete resolution
of their
network
would be necessary
for the appropriate
diet
guideline
to avoid
tional viewpoint.
calcium
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