Expression of intestinal brush-border membrane hydrolases and
ferritin after segmental ischemia-reperfusion in
rats
Kwo-Yih
Yeh,
Mary
Yeh, and
Jonathan
Glass
Departments of Medicine and Molecular and Cellular Physiology, and
Feist-Weiller Cancer Center, Louisiana State University Medical
Center, Shreveport, Louisiana 71130
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ABSTRACT |
Jejunal expression of three brush-border
membrane (BBM) enzymes, intestinal alkaline phosphatase (IAP),
lactose-phlorizin hydrolase (LPH), and sucrase-isomaltase (SI), and a
cytosolic protein, ferritin (Ft), was investigated after transient
segmental ischemia-reperfusion (I/R). I/R reduced mucosal IAP,
LPH, and SI mRNAs to 36%, 11%, and 38% of normal jejunal levels
after 3 h of reperfusion and to 22%, 8%, and 51% of normal jejunal
levels after 6 h of reperfusion, respectively. Intriguingly, in the
internal control jejunum IAP and LPH mRNAs also decreased
significantly. LPH and SI mRNA rapidly recovered to levels
significantly higher than those of normal jejunum at 12 h, whereas IAP
mRNA levels did not recover until 48 h. Enzyme activity paralleled
changes in mRNA levels in the ischemic reperfused jejunum.
Electrophoretic mobility shift assays showed that I/R significantly
increased SI footprinting 1 (SIF1) binding activity. The mobility of
one of the DNA-protein complexes was further retarded in the presence of anti-Cdx-2 antibody, suggesting that either Cdx-2 or a related protein was interacting with the SIF1 sequences. Similar to BBM enzymes, cytosolic Ft mRNA and protein were significantly decreased at
3 and 6 h after I/R. By 12 h, Ft mRNA, but not Ft protein, had
increased to higher than normal levels. We conclude that a rapid
recovery of BBM mRNAs and enzymes occurs in regenerating mucosa after
upper villus damage. The increase of SIF1 binding protein activity
after I/R may enhance SI, and perhaps LPH, gene transcription. The
expression of Ft is regulated at both pretranslational and
translational levels.
injury and repair; enterocyte differentiation; mRNA levels; nuclear
transcription factor; transcription and translation regulation
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INTRODUCTION |
THE SMALL INTESTINAL epithelium undergoes constant
renewal. New epithelial cells are produced in the crypts. The majority of new cells migrate from the crypt to the villus and undergo apoptosis
at the villus tip. The life span of rat absorptive cells from birth to
death is about 2-3 days (36, 46). During upward migration along
the villus column, cells express specific genes to perform digestive
and absorptive functions. Studies of epithelial cell differentiation
using important functional markers, such as lactose-phlorizin hydrolase
(LPH) and sucrase-isomaltase (SI), have shown that the expression of
these enzymes takes place in a temporal and spatial manner during the
cell life span (36, 40, 49). However, little information is available
on the molecular mechanisms by which epithelial cell differentiation is
regulated.
An approach to the analysis of regulatory mechanisms for
differentiation is to induce damage and exfoliation of differentiated villus cells and then to investigate during recovery the molecular processes leading to the differentiation of incompletely differentiated or newly generated cells. These injury and repair processes can be
produced by occlusion of intestinal blood flow for a short period
followed by reperfusion. An episode of ischemia-reperfusion (I/R) is known to provoke sequential events beginning with desquamation of villus cells, followed by a transient increase of crypt cell production and finally the migration and differentiation of the replenished villus cells for functional recovery (4, 12, 29, 35).
Specific gene expression during recovery from I/R would allow for the
analysis of the processes and regulatory mechanisms of cell
differentiation. We have used this experimental model to examine
absorptive cell expression of brush-border membrane (BBM) integral
proteins, such as intestinal alkaline phosphatase (IAP), LPH, and SI.
In this study, changes in IAP, LPH, and SI mRNA levels and enzyme
activities after I/R have been measured to determine whether each BBM
enzyme recovers in coordination with its encoding mRNA and whether the
rate of recovery is similar for each enzyme. The results show that the
activity of each enzyme recovers in parallel with the encoding mRNA but
that the time for recovery differs between enzymes. The coordinated
changes in transcript abundance and enzyme activity suggest that the
regulation occurs either by increased transcription and/or by
mRNA stabilization.
Among the BBM enzymes studied in this report, SI is thought to be
regulated primarily at the transcriptional level (17). Recently, an
evolutionarily conserved promoter element of the SI gene, SI
footprinting 1 (SIF1), has been identified in mice and humans. The
binding of SIF1 to a homeodomain protein (Cdx-2) is required for
promoter activity (37, 38). The presence of an SIF1 binding protein not
related to Cdx-2 has been reported in the intestine expressing SI in
adult rats (15). The possible regulatory role of SIF1 binding proteins
on SI expression under physiological conditions in vivo has not been
examined. To test the possibility that SIF1 protein is related to the
upregulation of SI expression, we examined the change of SIF1 binding
activity during recovery after I/R. We reasoned that if the SIF1
binding protein is involved in the upregulation of SI expression, an
increase of SIF1 activity might occur.
To examine whether the recovery of BBM enzymes is a phenomenon specific
to membrane proteins, we determined the expression of the cytosolic
protein ferritin (Ft) after I/R. Ft was chosen for the study because
1) similar to SI, it exhibits a
pattern of increasing expression as cells migrate upward along the
villi (11, 50); 2) it sequesters
intracellular iron and plays an important protective role against
oxidative stress-induced cellular damage after I/R (14, 16);
3) Ft expression is regulated
predominantly at the translational level by iron regulatory proteins
(IRPs) (14, 16), in contrast to the transcriptional regulation of SI
(16); and 4) both the IRP and SIF1
binding proteins are redox sensitive (25-27, 37).
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MATERIALS AND METHODS |
Animals and surgical induction of segmental intestinal I/R.
Twenty-one Sprague-Dawley rats, bred and raised in our animal quarters
and weighing 200-250 g, were used. The rats were fed overnight
with 5% glucose saline solution, anesthetized by administration of 50 mg/kg ip pentobarbital sodium, and subjected to laparotomy to
exteriorize the small intestine from the ligament of Treitz to the
ileocecal valve. Three rats were killed without further manipulation,
and 10-cm jejunal segments 15 cm below the ligament of Treitz were
collected as normal jejunum to serve as the external control. In the
other animals, a 10-cm jejunal loop beginning at 20 cm below the
ligament of Treitz was marked with two silk-thread ligatures, which
also ligated the arterioles and venuoles in the mesenteric margin of
the intestine to interrupt collateral circulation. To induce an episode
of segmental I/R within the jejunum, the blood flow of mesenteric blood
vessels connecting to the marked jejunal loop was interrupted by
clamping for 30 min (ischemia) and then released for blood
recirculation (reperfusion). After the induction of I/R, the small
intestine was returned to the peritoneal cavity and the wound was
closed in two layers with 3-0 silk sutures. The rats were provided with
5% glucose solution ad libitum and were killed at 3, 6, 11, 24, and 48 h after reperfusion. The I/R-injured jejunal loop, 0.5 cm away from
proximal and distal end markers, was removed and flushed with 10 ml of
ice-cold saline. The jejunal mucosa was gently scraped and collected
for biochemical assays and for total RNA isolation. For internal
controls, the mucosa of a 10-cm jejunal segment just above the
ischemic reperfused jejunum (I/R jejunum) was also collected
and designated as control jejunum.
To determine whether SIF1 activity is increased during recovery, an
experiment using 20 rats was performed. The rats were fed
5% glucose saline overnight and separated into two groups. The normal
controls (4 rats) were killed immediately after anesthesia, and the I/R
rats (16 rats) were subjected to segmental I/R and killed at 1, 3, 6, and 12 h after reperfusion (4 rats per each time point). In a separate
study, rats were subjected to segmental I/R and killed 24 h later. The
mucosal nuclear extracts were collected from the normal jejunum,
control jejunum, and I/R jejunum and were used for determination of
SIF1 binding activity by electrophoretic mobility shift assay (EMSA).
Indirect immunofluorescent staining of SI and LPH.
Digestive functional recovery depends on the expression of functional
proteins at an appropriate cellular location. Intestinal SI and LPH are
expressed in the BBM of villus but not crypt cells (36). To determine
the expression and cellular location of these enzymes, we used indirect
immunofluorescent staining methods with monospecific rabbit anti-rat SI
or LPH antiserum as the probe (48). In this study, a 1-cm segment at
the center of the I/R jejunum and a distal segment of control jejunum
were cut open, laid on a paraffin block, and fixed with 3%
paraformaldehyde-1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4)
at 4°C for 30 min. The fixed tissues were washed in phosphate
buffer, transferred to a 20% sucrose-phosphate buffer solution for 20 min, embedded in O.C.T. compound, frozen, and sectioned at
5-µm thickness. Sections from tissues at different time points were
mounted on the same slide to ensure that all tissues were stained under
the same conditions. The slides were examined as described previously
(48). Briefly, tissue sections were
1) washed with PBS;
2) preincubated with 10% control
rabbit serum at room temperature for 10 min, washed once with PBS, and
incubated with rabbit antiserum against rat SI or LPH (1:500 dilution)
for 1 h; 3) washed three times with PBS, 10 min each; 4) incubated with
FITC-conjugated goat-IgG anti-rabbit IgG (1:250 dilution) for 30 min;
5) washed three times with PBS; and
6) mounted in
Immunomount and observed under a Nikon microscope with an
epifluorescence attachment.
Biochemical assays and Western blots.
IAP, SI, and LPH activities were measured using the substrates
phenylphosphate, sucrose, and lactose as described previously (48, 49,
52). Protein content was measured by the method of Bradford (5).
Changes in the mucosal Ft content were detected by Western blot
analysis. Aliquots of mucosal homogenates containing 10 µg of protein
were mixed with the same volume of 2× sample buffer consisting of
120 mM Tris buffer (pH 6.8), 2% SDS, 20% glycerol, and 10%
2-mercaptoethanol, boiled for 5 min, and subjected to SDS-PAGE and
Western blotting as described previously (52). The monospecific rabbit
antiserum against rat Ft that was previously described (47) was used as
the probe, which was detected by an enhanced chemiluminescence Western
blotting detection kit according to the manufacturer's directions
(Amersham, Arlington Heights, IL). The signal was quantitated by
transmittance densitometry, using volume integration with ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Northern blot analysis.
Mucosal RNA was isolated according to the method of Chomczynski and
Sacchi (10).
The quantity of total RNA was determined by absorbance at 260 nm. Total
RNA was size fractionated by formaldehyde gel electrophoresis and
vacuum transferred to a Nylon membrane (Hybond-N; Amersham). After
blotting, RNA was cross-linked to the membrane in a GS Gene Linker
Ultraviolet Chamber (Bio-Rad, Hercules, CA).
Labeled cDNA probes were prepared by random primer labeling with
[32P]dCTP using a
Prime-It labeling kit (Stratagene, La Jolla, CA). The cDNA probes were
gel purified after restriction digestion of the rat IAP-I (21), LPH
(6), or SI cDNA (39) clones, which were generously provided by Drs.
David Alpers, Richard Grand, and Peter Traber, respectively. For the
analysis of intestinal Ft expression, the rat duodenal ferritin
heavy-chain (Ft H) cDNA was cloned using the standard PCR
technique. The cDNA beginning at base +196 to the last base of Ft H was
amplified by PCR with oligonucleotide primers (5' primer:
5'-C
CGCGTCTCCCTCGCA-3'; 3' primer:
5'-
AAAATTCTTTAT-3'),
which were synthesized according to the sequence reported by Murray et
al. (24). The 5' primer had an
EcoR I site and the 3' primer a
Kpn I site (underlined). The 700-bp
nucleotide produced by PCR was cloned using the pCR-Script SK(+)
cloning kit (Stratagene). The nucleotide sequence of the cDNA was 99%
identical to the published hepatic Ft H cDNA sequence (24). To
normalize the quantity of RNA samples applied to the gels, the amount
of 28S rRNA in each sample was determined using a
32P-labeled riboprobe prepared by
in vitro transcription reaction with a linearized rat 28S rRNA clone.
Hybridization was performed using QuikHyb according to the
manufacturer's instructions (Stratagene). After hybridization, the
membrane was washed three times for 10 min each in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% SDS at room temperature and twice for 15 min each in 0.1×
SSC-0.1% SDS at 65°C. The radioactivity was detected using a
Phosphorimager and quantitated with ImageQuant software as described
previously (47).
Preparation of nuclear extracts.
For the determination of SIF1 activity, nuclear extracts were prepared
from isolated nuclei. The nuclei were isolated by a modified method
described by Lamers et al. (20). Briefly, mucosal scrape (~200 mg)
was washed with 20 ml ice-cold PBS, suspended in 10 ml of cell lysis
buffer consisting of 10 mM HEPES (pH 7.5), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), 200 mM sucrose, 0.5% Nonidet P-40 (NP-40), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, and 1 µg/ml aprotinin, homogenized by 60-80 strokes in a Dounce
homogenizer with pestle B to obtain >80% lysed cells (monitored by
phase-contrast microscopy), filtered through four layers of
cheesecloth, and centrifuged at 800 g
for 5 min to obtain the crude nuclear pellet. The pellet was
resuspended in 25 ml of cell lysis buffer without NP-40 but containing
1.65 M sucrose. The crude nuclear suspension was layered on a sucrose cushion solution (2 M sucrose, 2 mM
MgCl2, and 10 mM
Tris · HCl, pH 7.5) and centrifuged 25,000 g for 1 h. The nuclear pellet was then
resuspended in 300 µl nuclear extract buffer (20 mM HEPES, pH 8.0, 20% glycerol, 0.1 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 1 µg/ml each of leupeptin and aprotinin), sonicated at 15% power
output for 5 s, and centrifuged at 12,000 g for 1 min. The nuclear extract was
transferred to fresh tubes and stored at
72°C in aliquots.
To examine the tissue specificity of SIF1 activity, two rats at 3 h
after I/R were anesthetized and subjected to left ventricle perfusion
with Ca2+- and
Mg2+-free Hanks' balanced salt
solution containing 30 mM EDTA as described by Bjerknes and Cheng (3).
After separation of the epithelium, the lamina propria was gently
scraped with glass slides to obtain presumably mesodermal cells. The
nuclear extract was then isolated from the epithelial and mesodermal
cells.
EMSA.
The DNA binding activity of SIF1 in the nuclear extract was determined
by EMSA. Oligonucleotides of the rat SIF1 element
(5'-TGTGAAAGTGCAATAAAACTTTATGAGTA-3' and
5'-TGACTACTCATAAAGTTTTATTGCAC-3') were synthesized
according to the rat nucleotide sequence reported by Hecht et al. (15) and labeled with
[32P]dATP with the use
of a filling reaction in the presence of a Klenow fragment of DNA
polymerase 1 to form a double-stranded SIF1 motif corresponding to base
pairs
62 to
29 from the transcriptional start site. The
labeled probes were separated from free
[32P]dATP on an NAP-5
column (Pharmacia Biotech, Piscataway, NJ). EMSA was performed in 15 µl containing 10-15 µg nuclear extract protein, 20 mM HEPES,
pH 7.5, 60 mM KCl, 10 mM MgCl2,
0.5 mM DTT, 1 mM EDTA, 12.5% glycerol, 1 µg poly(dI-dC), and 1 × 105 counts per minute
32P-labeled DNA. The mixture was
incubated at room temperature for 30 min and analyzed by
electrophoresis on a 5% nondenaturing polyacrylamide gel, using
high-iron conditions described by Ausubel et al. (1). To examine
whether the protein-DNA complexes contained the SIF1 binding factor
Cdx-2, supershift assays were performed using affinity-purified antibody against mouse Cdx-2 (kindly provided by Dr. Peter R. Traber)
(38).
Statistical analysis.
ANOVA was computed from the experimental data. When the
F value obtained from ANOVA was
significant, Bonferroni's test was applied to test for differences
among groups. When comparing values of I/R jejunum and control jejunum
in the same animal, paired Student's
t-test was used.
P < 0.05 was considered significant.
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RESULTS |
Changes in the intestinal mucosal abundance of IAP, LPH, SI, and Ft
H transcripts after I/R.
Figure 1 shows the changes in the abundance
of IAP, LPH, SI, and Ft H transcripts in normal jejunum, internal
control jejunum, and I/R jejunum after reperfusion by Northern blot
analysis. All transcripts were significantly decreased in I/R jejunum
after 3 and 6 h of reperfusion (Figs. 1 and
2). Local segmental I/R also produced a
modest reduction in all transcripts in the control jejunum; the
decrease was significant for IAP and LPH mRNAs, but not SI and Ft H
mRNAs, compared with the mRNAs of the normal jejunum (Figs. 1 and 2).
The recovery of the transcripts in I/R jejunum occurred to different
extents and at different times: LPH, SI, and Ft H mRNA levels had
recovered and increased to levels ~50% higher than normal levels at
12 h of reperfusion (P < 0.05 for all transcripts), whereas IAP mRNA did not recover until 48 h (Figs. 1
and 2). By 24 h of reperfusion, the LPH and Ft H mRNAs had subsided,
whereas SI mRNA increased further and then decreased to a level still
higher than that of normal jejunum at 48 h (Fig. 2). The overshoot
phenomenon of mRNA during recovery also occurred in control jejunum, in
which LPH and SI mRNA increased to a level higher than that in normal
jejunum at 12 and 24 h, respectively (Fig. 2).

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Fig. 1.
Northern blot analysis of intestinal alkaline phosphatase (IAP),
lactose-phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and
ferritin heavy-chain (Ft H) mRNA and 28S rRNA in intact normal jejunum
(N-J), internal control jejunum (C-J), and ischemic reperfused
jejunum (I/R-J). Rats were fed a glucose solution overnight and were
either killed (control) or subjected to surgical induction of jejunal
segmental ischemia for 30 min, followed by reperfusion for
recovery. Animals were killed at indicated times after reperfusion, and
the ischemia-reperfusion (I/R) segment and the control segment
(immediately above the I/R segment) were collected for analysis (see
MATERIALS AND METHODS for details).
About 10 µg total RNA per sample was size separated by
electrophoresis in a 1% formaldehyde agarose gel and transferred to a
Nylon membrane. The same membrane was sequentially blotted with
32P-labeled probes, and the probe
was removed by stripping after each blot. All mRNAs were detected by a
storage Phosphorimaging system. Reduction of all mRNAs in I/R-J was
noted at 3 and 6 h. Restoration of mRNA levels occurred at different
times after reperfusion for each mRNA. 28S rRNA showed that equivalent
amounts of RNA samples were applied to each lane.
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Fig. 2.
Effect of I/R on the abundance of mucosal IAP, LPH, SI, and Ft H
transcripts. Radioactive signals from Northern blots as shown in Fig. 1
were quantitated by volume integration using a Phosphorimaging system
with ImageQuant application software (see MATERIALS
AND METHODS for details) and were normalized to 28S
rRNA in each sample. Data are means ± SE, expressed as percentage
of normal levels (n = 3-4). + P < 0.05 and
++ P < 0.01 vs. C-J;
* P < 0.5 and
** P < 0.01 vs. N-J.
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Changes in villus height and mucosal IAP, SI, and LPH activities and
Ft content after I/R.
Figure 3 shows morphological changes in the
intestine after I/R, resulting in a significant decrease in villus
height (Fig. 4). Intestinal damage that
occurred at 1 h after reperfusion was readily observed at the boundary
between control jejunum and I/R jejunum (Fig.
3A). The control jejunum (Fig.
3A,
right) showed intact villi, whereas
the I/R jejunum (Fig. 3A,
left) showed upper villus cells
extruding into the lumen (Fig. 3A).
By 3 h, the I/R jejunum had lost most upper villus cells and showed
significantly decreased villus height (Figs.
3B and 4). The villus height did not
increase until 24 h after reperfusion (Figs.
3C and 4) and had recovered to normal
levels at 48 h (Figs. 3D and 4). In
contrast to the villus height, the crypt depth showed a statistically
insignificant trend of increasing rather than decreasing depth after
reperfusion (data not shown).

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Fig. 3.
Micrographs of intestinal mucosa after I/R (bar = 25 µm).
A: boundary between I/R-J
(left) and C-J
(right) 1 h after reperfusion. Upper
villus cells were extruded in the area of I/R-J, whereas villus
remained intact in C-J. B,
C, and
D: I/R-J at 3 (B), 24 (C), and 48 h
(D) after reperfusion, respectively.
Villus height decreased ~60% at 3 h, partially recovered at 24 h,
and fully recovered by 48 h.
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Fig. 4.
Effect of I/R on villus height. Villus height was measured from villus
tip to crypt-villus junction under ×200 magnification. Data are
means ± SE (n = 3-4).
P < 0.01 and
* P < 0.05 vs. control
jejunum.
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IAP, sucrase, and lactase specific activity decreased significantly in
I/R jejunum at 3 and 6 h after reperfusion (Fig.
5). The reduction of enzyme activity was
less than the reduction of the corresponding mRNA; both the IAP and LPH
specific activities showed 50% decreases, compared with a 78%
reduction in IAP mRNA and a 92% decrease in LPH mRNA at 6 h of
reperfusion (Figs. 2 and 5, control jejunum vs. I/R jejunum).
Similarly, SI activity decreased 40% compared with a 62% reduction in
SI mRNA at 3 h after reperfusion. In the control jejunum the specific
activity of all three enzymes was not affected by distal jejunal I/R,
though IAP and LPH mRNA both decreased significantly. Because the
mucosal weight was decreased after I/R (Table
1) and the tissue exfoliated into the lumen
consisted of upper villus cells expressing high levels of BBM enzymes,
the total activity (expressed as mU/cm intestine) decreased more than
the specific activity (Table 1). At 12 h of reperfusion, both the LPH
and SI specific activity had recovered to normal levels, whereas the
total activity was still lower than the normal level because the
mucosal weight remained significantly lower (Table 1). In contrast to
LPH and SI, IAP specific and total activity did not return to normal
levels until 48 h of reperfusion (Table 1).

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Fig. 5.
Effect of segmental jejunal I/R on IAP, lactase (L), and sucrase (S)
enzyme activity at 3, 6, 12, 24, and 48 h after reperfusion. Enzyme
units (U) are expressed as micromoles of substrate hydrolyzed per
milligram protein per minute. Data are means ± SE
(n = 3-4). Dashed line across
each panel represents mean enzyme activity of N-J. + P < 0.01 and
++ P < 0.05 vs. C-J;
* P < 0.05 and
** P < 0.01 vs. I/R-J at
immediate earlier time point.
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The mucosal Ft content in the I/R jejunum showed a reduction of 40% at
1 h and 65% at 6 h and then partially recovered at 12 h after I/R
(Fig. 6). In the control jejunum, the
mucosal Ft content did not change at 1 h but decreased significantly at
3 and 6 h and recovered partially at 12 h after I/R (Fig. 6,
A and B). The decrease of mucosal Ft
protein in the control jejunum was in contrast to the three BBM
enzymes, whose activity did not decrease. The decrease of Ft protein
that occurred in the absence of a coordinated decrease of Ft mRNA
levels in the control jejunum suggests that either translational
repression and/or increased Ft protein degradation had
occurred.

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Fig. 6.
Changes in Ft protein after I/R determined by Western blots.
A: representative Western blot
analysis of Ft in mucosa of N-J, C-J, and I/R-J. Aliquots of mucosal
homogenates containing 10 µg protein were subjected to SDS-PAGE under
denaturing conditions, the protein was transferred to a nitrocellulose
membrane, and Ft was detected by rabbit antiserum to rat Ft, using the
enhanced chemiluminescence blot kit (see MATERIALS AND
METHODS for details).
B: Ft protein was quantitated by
transmittance densitometry, using volume integration with ImageQuant
application software. Data are expressed as percentage of Ft in the N-J
(means ± SE, n = 3).
* P < 0.05 vs. C-J;
§ P < 0.05 vs. immediate
earlier time point.
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Localization of immunoreactive SI and LPH.
The normal jejunum showed a pattern of gradual increase in SI
expression along the villus axis (Fig.
7A). SI was undetectable in the
crypts, appeared at the villus base, and increased to plateau in the
upper fourth of the villus (Fig.
7A). The
SI was predominantly located at the BBM (Fig.
7A). In samples stained with control antiserum, the BBM was devoid of immunoreactivity, whereas some of the
undefined nonepithelial cells showed nonspecific fluorescent staining
(not shown). I/R did not alter subcellular localization of SI but
markedly decreased the villus height and reduced BBM immunoreactivity
at 3 or 6 h after I/R (Fig. 7C). The
exfoliated cells remaining in the lumen of I/R jejunum showed positive
fluorescent staining (Fig. 7C). By
12 h, immunoreactivity was detected not only in BBM of villus cells but
also at the apical surface of cells in the crypt bottom (Fig.
7D). By 24 h, SI immunoreactivity in
the crypt cells had subsided (Fig.
7E). By 48 h, I/R jejunum had
completely recovered villus height, with the villi covered by
epithelial cells showing increased SI immunoreactivity throughout the
BBM of villus cells (Fig. 7F). It is
worth noting that upregulation of SI expression might occur in cells at
the villus base, as these cells had levels of SI immunoreactivity
comparable to those at the upper villus (Fig.
4F). LPH was also localized at the
BBM of the villus cells. Changes in LPH immunoreactivity after
reperfusion were the same as those of SI, except there was no
fluorescent stainability in the BBM of the crypt cells at 12 h after
reperfusion (data not shown).

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Fig. 7.
Localization of SI expression by indirect immunofluorescent staining
(bar shown in A = 35 µm for
A, B,
C, E,
and F and 100 µm for
D).
A: normal jejunum. Immunoreactive SI
is localized in brush-border membrane (BBM; arrows) of absorptive cells
with increasing immunofluorescence from villus base to a plateau at the
upper fourth of the villi. Continuity of the immunoreactive BBM is
interrupted by goblet cells (arrowheads). Unidentified leukocytes in
the villus core show nonspecific fluorescent staining.
B: control jejunum 6 h after distal
segmental I/R. Villus height and vertical gradient of immunoreactive SI
at the BBM are similar to those of normal jejunum.
C: I/R-J at 6 h after I/R. Compared
with control jejunum, I/R-J shows smaller and shorter villi, which are
lined with epithelial cells showing weak SI staining at BBM. Some
exfoliated upper villus cells in the lumen retain immunoreactivity.
D: I/R-J at 12 h after I/R.
Immunoreactive SI is detected not only in the BBM of the villus
absorptive cells (arrows) but also in cells located at the crypt base
(arrowheads). No fluorescent activity is detected in goblet cells.
E: I/R-J at 24 h after I/R. Villi are
still short but are covered by the epithelium with strongly stained
fluorescent BBM. Cells at base of crypts show no detectable
immunoreactive SI. F: I/R-J at 48 h
after I/R. Villi have recovered to normal height and are lined by
epithelium expressing higher levels of SI at the BBM throughout the
villus. High fluorescent intensity is consistent with high specific and
total activity measured biochemically.
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Changes in SIF1 activity.
Figure 8 shows that SIF1 binding proteins
in the nuclear extract from the mucosa of normal jejunum and control
jejunum produced two major protein-DNA complexes, A and B, confirming
an earlier report (37). In addition, there were faint complexes, C and D, with greater mobility. SIF1 binding activity was present at low
levels in both normal and control jejunum (Fig.
8A) and was significantly increased
in the I/R jejunum throughout the first 12 h of reperfusion (Fig. 8).
Interestingly, complexes A-D showed specific temporal patterns after
reperfusion: the increase of complex A did not occur until 6 and 12 h;
the increase of B was detected at 1 h and continued during the 12-h
period (Fig. 8A); complex C
increased from 1 to 6 h and decreased to a low level by 12 h; and
complex D increased at 1 h and decreased quickly to the background
level. Complexes C and D were reproducibly detected in our laboratory.
It is possible that the proteins in C and D complexes might be the
degraded products of activated proteins in A and B complexes after
tissue injury in I/R jejunum, as complexes C and D were not present in
the nuclear extract of I/R jejunum at 12 h. The SIF1 protein binding
was specific, as all complexes were not detected when a 100-fold
concentration of competitor DNA was included in the assay (Fig.
8A). Incubation of the nuclear extract with rabbit anti-mouse Cdx-2 produced a supershift band with a
concomitant decrease and/or disappearance of complex A in
control jejunum and I/R jejunum at 1 h and in I/R jejunum at 12 h after
reperfusion (Fig. 8A, last 3 lanes).
The supershift indicates that Cdx-2 or a related protein participated
in the formation of complex A. SIF1 binding activity was present in the nuclear extract of epithelial cells but not submucosal cells (data not
shown).

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|
Fig. 8.
Electromobility shift assay (EMSA) of SI footprinting 1 (SIF1) protein
binding activity in jejunal nuclear extracts.
A: rats were fed 5% glucose saline
overnight and subjected to surgical induction of segmental I/R. Intact
rats were killed after anesthesia to serve as normal controls. The
mucosal scrape was collected from normal jejunum (N-J), internal
control jejunum (C-J), and ischemic reperfused jejunum (I/R-J)
for isolation of the nuclear extract. An aliquot of nuclear extract
containing 15 µg protein was used for each assay. Low levels of SIF1
binding activity were observed in A,
B, C,
and D bands in N-J and C-J. These four
bands became conspicuous in I/R-J 1 h after reperfusion, and each
complex showed specific temporal changes during the first 12 h after
reperfusion. The DNA-protein interaction was not detected when 100-fold
competitor was included in the assay. Incubation of nuclear extract
from 1 h C-J and 1 and 12 h I/R-J with polyclonal antibodies against
Cdx-2 for 30 min produced a supershift band in association with the
decrease of complex A (last 3 lanes).
B: changes in SIF1 binding activity in
C-J and I/R-J. Radioactive signals of A and B complexes, as shown in
Fig. 8A, were detected by a storage
Phosphorimaging system and quantitated with ImageQuant software. Data
are means ± SE (n = 4).
Significant increases in the activity of SIF1 binding protein occurred
in I/R-J at 1 h, and this increase was retained during the 12 h of the
experiment. * P < 0.01 vs.
C-J.
|
|
Because A and B complexes have been shown to be specific
for SIF1 DNA binding in Caco-2 cells (40) and they were the only complexes detected in the I/R jejunum at 12 h after reperfusion, the
radioactivity in the two complexes was added to quantitate SIF1
activity (Fig. 8B). SIF1 activity
was rapidly activated after I/R, and the high levels of activity were
sustained throughout the first 12 h after reperfusion (Fig.
8B). By 24 h, SIF1 activity had
largely subsided but was still significantly higher in the I/R jejunum
than in control jejunum (not shown).
 |
DISCUSSION |
The mechanisms by which temporal and/or spatial regulation of
cell differentiation occurs along the crypt-villus axis during the
short 2- to 3-day life span of enterocytes are poorly understood. The
present studies were undertaken to examine molecular events occurring
as the regenerated and/or immature cells differentiate after
the loss of upper villus cells induced by I/R. A short 30-min period of
ischemia with subsequent reperfusion has been reported to cause
upper villus cell exfoliation (4). A prolonged 90-min period of
ischemia followed by reperfusion destroys the villus with a
90% decrease in the activity of disaccharidases (18). We observed that
30 min of ischemia followed by reperfusion caused a 60%
decrease in the specific activity of IAP and 40-50% reduction in
both lactase and sucrase activities at 3 and 6 h. The decrease was not
limited to the BBM enzymes, as cytosolic Ft showed a 40-65% decrease at 1-6 h after I/R. The decrease of the BBM proteins and
Ft appears to be primarily the result of premature exfoliation of upper
villus cells, as the shortened villi were covered by the epithelium
expressing low levels of SI immunoreactivity. The decrease in mRNA
levels would not be predicted from the usual mRNA distribution along
the villus axis; as IAP, LPH, and SI transcripts are higher in the
lower than in the upper villus (38, 39, 45), the loss of upper villus
cells should cause a greater decrease in enzyme activity than in mRNA
levels. The present observation that after I/R BBM mRNAs were reduced,
in some instances to a greater degree than the reduction of enzyme
activities (Figs. 2 and 3), suggests increased mRNA degradation after
I/R.
A significant decrease of Ft, but not BBM enzymes, was found in the
control jejunum at 3 and 6 h after I/R of distal jejunum. Since the
precocious exfoliation of upper villus cells did not occur in the
control jejunum, the decrease of Ft must have resulted either from a
decrease in Ft synthesis, an increase in Ft degradation, or both. The
decrease of Ft content without a parallel decrease in the Ft mRNA
levels suggests either translational repression of Ft mRNA or
accelerated Ft protein degradation. Direct identification of the actual
mechanism will depend on measurements of Ft synthetic and degradative
rates. It is well established that Ft synthesis is negatively regulated
at the translational level through the binding of IRP to the IRE on the
5' untranslated region of the Ft mRNA (14, 16). The increase of
IRP activity has been reported in cultured cells under oxidative
stress, resulting in the suppression of Ft mRNA translation (16,
25-27). The decrease of Ft mRNA translation in the control jejunum
may well be related to the oxidative stress induced by I/R in the more
distal jejunal segment. The phenomenon of remote tissue damage after
intestinal I/R has been reported in the lung and liver (8, 9, 13) and
is thought to be mediated by activated leukocytes and inflammatory
cytokines in the circulation (8, 9, 13, 28, 34). The precise mechanism by which a circulatory mediator causes translational repression of Ft
mRNA in the control jejunum is unknown. Physiologically significant
levels of tumor necrosis factor-
(TNF-
) and interleukin-1
(IL-1
) have been reported to be released from human intestinal segments after 30 min of ischemia followed by reperfusion (44). Moreover, intestinal I/R has been reported to elevate plasma
concentrations of TNF-
, IL-1
, IL-6, and endotoxin (9, 28). It is
likely that these circulatory cytokines released from the I/R jejunum enhance expression of adhesive molecules in the endothelium of remote
organs (8, 13, 23). Subse- quently, these adhesive molecules would
recruit and activate circulating leukocytes to peripheral tissues (23,
28). In our experimental model, activated macrophages and/or
neutrophils that lodged in the mucosa of control jejunum may release
hydrogen peroxide and nitric oxide, which subsequently activate IRP and
repress Ft synthesis (16, 25-27). This distant tissue effect takes
place later than in the local tissues, as the decrease of Ft protein
was detected at 3 h in control jejunum compared with 1 h in I/R
jejunum. The indirect tissue effects might also have caused the
reduction the IAP, LPH, and SI mRNAs in the control jejunum. In Caco-2
cells, cytokines such as IL-6 and interferon-
have been reported to
decrease SI but not LPH synthesis, and the magnitude of the
downregulation is greater than that of upregulation by TNF-
(53).
Thus segmental intestinal I/R elicited effects on the expression of
diverse genes in the local and distant enterocytes; the effects were
both gene specific and occurred at different levels of regulation.
The LPH and SI activity and immunoreactive SI recovered quickly, by 12 h after I/R. The increase of LPH and SI expression occurred in the
absence of an appreciable increase in the mucosal weight, a phenomenon
consistent with earlier reports that the recovery of villus cellularity
is independent of BBM hydrolase or functional recovery (12, 18, 31).
Our data showed that LPH and SI enzyme recovery occurred before, rather
than after, the restoration of villus cellularity (18, 31). Differences in the depth of mucosal damage might account for this variance from
earlier reports, as the duration of ischemia was 30 min in the
present study and 90-180 min in other studies (18, 31).
Although changes in SI immunoreactivity observed with indirect
immunofluorescent staining methods might not reflect tissue SI content,
the observation of parallel changes in SI immunoreactivity and sucrase
activity measured by biochemical assay after I/R implies a quantitative
increase in SI. These parallel changes are particularly conspicuous in
48 h I/R jejunum, in which the BBM throughout the villus showed high SI
immunoreactivity (Fig. 4F) and
intestinal SI activity was significantly higher than normal (Fig. 2 and
Table 1). The presence of immunoreactivity
in the BBM of the crypt bottom cells at 12 h after I/R,
but not later, suggests a transient activation of the SI gene that is
normally not expressed in the crypts. Whether the activation of the SI
gene in the crypts is related to a local increase in SIF1 binding
activity remains to be determined.
The expression of LPH and SI, and perhaps IAP, is known to be regulated
essentially at the level of transcription (17). The present study found
that the recovery of IAP, LPH, and SI mRNAs and their protein products
occurred in parallel, agreeing with transcriptional regulation. It is
worth noting that BBM mRNAs did not recover in parallel, as lactase and
sucrase returned to normal levels at 12 h, whereas IAP did not return
to normal levels until 48 h after I/R, indicating that each gene is
differentially regulated. The concomitant recovery of LPH and SI
appears to be more than coincidental. A
cis-element conferring positive
regulation for LPH expression contains the nucleotide sequence TTTAT
(NF-LPH1), which is very similar to the
cis-element with a palindromic SIF1 sequence TTTAC (37, 38, 42). Both sequences have been demonstrated to
compete for binding to Cdx-2 or nuclear factors closely related to
Cdx-2 (41). The significant increase in the activity of SIF1 binding
protein detected in the I/R jejunum might be related to the
upregulation of both LPH and SI expression during recovery after I/R. A
recent report suggests that the rat intestinal SIF1 binding protein
might not be related to Cdx-2 (15). However, the present EMSA
demonstrated that the antibody against mouse Cdx-2 produced a
supershift band with the disappearance of complex A (Fig.
8A). These data provide evidence
indicating that the SIF1 binding protein in complex A of the rat
intestinal nuclear extract is either Cdx-2 or a Cdx-2-related protein,
consistent with earlier findings in mouse, pig, and human intestine
(37-42). Complexes B and C shown in Fig.
8A might be equivalent to complexes IV
and III reported in adult rats (15), as the anti-Cdx-2 antibodies altered neither mobility nor activity of these complexes. The identity
of complexes B and C, as well as D, remains to be defined.
In addition to SIF1 binding and/or Cdx-2 proteins, other
transacting factors might also be involved in the upregulation of SI
expression, since a significant increase in the SI mRNA, but not LPH
mRNA, occurred in the control jejunum at 24 h without an increase of
SIF1 binding activity (Figs. 2 and 8). Recently, a consensus binding
site for GATA zinc finger transcription factors, in addition to the
SIF1 site, has been identified as necessary for transcriptional
activation of SI (35). Thus it is possible that the increase of an
undefined GATA protein occurred in control jejunum, resulting in the
increase of SI expression.
Similar to BBM proteins, mucosal Ft mRNA increased to higher than
normal levels at 12 h after I/R (Fig. 2). This recovery may reflect an
increase of Ft H gene transcription, probably mediated by oxidative
stress activation of nuclear transcription factor-
B (NF-
B), which
has been reported to enhance Ft gene expression in response to TNF-1
in cultured mouse fibroblasts (19). Our recent preliminary data showed
that NF-
B is activated in I/R jejunum prior to the recovery of Ft,
although it is not known whether the rat Ft H gene contains
B
enhancer elements (51). Alternatively, increased Ft H gene
transcription may be directly related to an increased intracellular
free iron pool after intracellular Ft degradation. It has been reported
that expansion of the intracellular free iron pool and increased Ft
expression occurred after oxidative stress in the liver (7). The
present data showing the recovery of Ft H mRNA, but not Ft protein, at
12 h after I/R indicate that the free iron pool in the I/R jejunum was
not high enough to totally derepress mRNA translation. Further study of
rat Ft H gene structure is needed to confirm the role of NF-
B in the
upregulation of Ft expression.
The rapid recovery of intestinal BBM enzymes and Ft mRNAs and mucosal
cellularity after I/R injury underscores the high adaptability of the
intestinal epithelial cells for regeneration and differentiation. The
recovery apparently depends on the genetic programs of surviving crypt
and lower villus cells. The present observation that in surviving cells
SIF1 binding protein and/or Cdx-2 activity was increased in
association with SI and perhaps LPH recovery suggests a mechanism of
transcriptional activation of SI and LPH genes. Recently,
overexpression of Cdx-2 in Caco-2 cells has been shown to stimulate SI
and LPH expression (22). Other genes related to cell survival and cell
proliferation must also be activated after I/R. In preliminary studies,
we observed that I/R increased NF-
B and activation protein-1 (AP-1)
activity (Ref. 51 and unpublished data); NF-
B has been reported to
play a role for cell survival (2, 43), and AP-1 activity is known as an
early response to growth stimulation (32, 33).
 |
ACKNOWLEDGEMENTS |
This study is supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Research Grants DK-37866, DK-41279,
and DK-43785 and by the Center for Excellence in Cancer Research,
Treatment, and Education, Louisiana State University Medical Center,
Shreveport, LA.
 |
FOOTNOTES |
Address for reprint requests: K.-Y. Yeh, Section of
Hematology/Oncology, Dept. of Medicine, Louisiana State Univ. Medical
Center, Shreveport, LA 71130.
Received 23 September 1997; accepted in final form 5 May 1998.
 |
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